Compositions and methods for determining likelihood of twinning

ABSTRACT

Collections of polynucleotides useful for estimating breeding value or detecting likelihood of twinning are disclosed. The polynucleotides are used to detect genomic sequences quantitatively associated with the twinning trait. Also disclosed are methods and kits for using the collections to estimate breeding value or predict likelihood of twinning.

CROSS-REFERENCE TO RELATED APPLICATIONS

This claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 61/100,697, filed Sep. 26, 2008, the entire contents of which are incorporated by reference herein.

GOVERNMENT RIGHTS

Pursuant to 35 U.S.C. §202 (c), it is acknowledged that the United States Government may have certain rights in any invention described herein, which was made in part with funds from the United States Department of Agriculture—National Research Initiative Grant No. 2005-35205-15556 and CREES Grant No. 08-CRHR-6055.

FIELD OF THE INVENTION

The invention described herein relates generally to animal genetics and improvements in cattle breeding. More particularly, it relates to compositions and methods for predicting the likelihood of twinning in cattle.

BACKGROUND OF THE INVENTION

The trait of twinning rate has been shown to possess economic value to the dairy industry. The negative effects of increased twinning rate include increased dystocia, retained placenta, longer return-to-estrus intervals, an increased frequency of freemartins, reduced milk production and more frequent involuntary culling (Beerepoot et al. 1992; Niellen et al. 1989). It has been estimated that economic loss to the dairy industry is around $110/cow due to lost revenue and increased costs from producing twin calves instead of singles (Beerepoot et al. 1992; Eddy et al. 1991); after accounting for effects of inflation, the cost in current dollars is likely considerably higher. Reducing the incidence rate of twinning will therefore reduce this loss and increase profitability for the dairy industry.

Genetic progress for selection on twinning rate has been made (Gregory et al. 1997). Non-zero estimates of heritability for the trait have been observed, suggesting an additive genetic component. Estimates have ranged from 0.06%-21.6% depending on parity inclusion and type of model used in its estimation (Johanson et al. 2001; Ron et al. 1990; Karlsen et al. 2000). Nevertheless, the majority of evidence suggests twinning rate to be a lowly heritable trait, and therefore an excellent candidate for the use of marker assisted selection to aid in its genetic progress.

Various studies have identified QTL affecting twinning rate or ovulation rate in cattle (Blattman et al. 1996, Kappes et al. 2000, Kirkpatrick et al. 2000, Lien et al. 2000, Arias and Kirkpatrick, 2004, Gonda et al. 2004, Cruickshank et al. 2004, Cobanoglu et al. 2005). Linkage mapping has shown positive results for the existence of QTL segregating in the Holstein population. A drawback of linkage mapping is that confidence intervals remain broad (Andersson & Georges 2004). In many reports throughout the literature, analyses have attempted to refine the locations of QTL using additional markers or larger datasets; however, this has been accomplished with minimal success. Alternative tools or approaches are necessary to refine these locations.

Recent advances in technology have facilitated screening of the genome with markers at higher density than previously possible (Hardenbol et al. 2005; Thompson et al. 2007). An example of a proposed analysis method has been reported by Meuwissen & Goddard (2001). Their method predicts identity by descent (IBD) probabilities at a given location using the information from the marker genotypes, or haplotype, surrounding the given location. Haplotypes provide more information compared to single-markers when linkage disequilibrium (LD) is weak, consequently improving the power of QTL detection.

Twinning or ovulation rate QTL on bovine chromosome 5 (BTA5) have been identified in previous studies. Twinning rate QTL were detected between 55-65 Mb (bovine genome assembly 3.1) on BTA5 in the Norwegian dairy cattle population (Lien et al. 2000) and the North American Holstein population (Cruickshank et al. 2004). An ovulation rate QTL was detected in the 40 cM (˜30 Mb) region of BTA5 (Kappes et al. 2000) in the USDA twinning population.

Though progress has been made, there remains a need for improved methods and genetic markers for predicting twinning potential for individual animals and within herds.

SUMMARY OF THE INVENTION

One aspect of the invention features a collection of one or more polynucleotides, each of which is at least partially complementary to a sequence in a bovine genome, the presence or absence of which such complementary sequences in the bovine genome is quantitatively associated with the trait of twinning in a cattle population, wherein the collection comprises at least one sequence that is quantitatively associated with twinning with statistical significance of at least p≦0.01. More particularly, the collection comprises at least one sequence that is quantitatively associated with twinning with statistical significance of at least p≦0.005, and even more preferably p≦0.001. In various embodiments, the collection is useful for estimating breeding value in cattle In certain embodiments, each polynucleotide represents one allele of a SNP in the bovine genome.

At least one of the polynucleotides is complementary to a sequence located on bovine chromosome 5 (BTA5). More specifically, at least one of the polynucleotides is complementary to a sequence that maps between 55-75 Mb of BTA5; or, more particularly, between 64-73 Mb of BTA5. In another embodiment, one or more of the polynucleotides is complementary to a sequence that maps on BTA5 at any of 24-26 Mb, 49-51 Mb, 64 to 65 Mb, or 69-70 Mb, or, more particularly, between 69.2 to 69.9 Mb.

At least one of the polynucleotides may be complementary to a sequence located in a genomic sequence for Insulin-like Growth Factor 1 (IGF1). Alternatively, at least one of the polynucleotides is complementary to a sequence in a genomic sequence for synapsin III (SYN3), synapsin III isoform A (SYN3A), collagen type X alpha-1 (COL10A1) precursor, eukaryotic translation initiation factor 3, subunit 7 (EIF3S7), insulin-like growth factor 1 (IGF1), inhibin beta-C (INHBC), cytoskeleton-associated protein 4 (CKAP4), or Alpha2-HS glycoprotein (AHSG.)

In various embodiments, the collection comprises at least one polynucleotide useful for detecting one or more particular SNPs of the bovine genome or BTA5, as described in detail herein.

Another aspect of the invention features a method for estimating the likelihood of twinning in one or more members of a cattle population comprising the steps of (a) providing a collection of one or more polynucleotides, each of which is at least partially complementary to a sequence in a bovine genome, comprising at least one sequence that is quantitatively associated with twinning with statistical significance of at least p≦0.01; (b) using the collection to determine the presence or absence of sequences complementary to one or more polynucleotides from the collection in one or more members of the cattle population genome, wherein the presence or absence of the complementary sequences is quantitatively associated with the trait of twinning in a cattle population; and (c) estimating the likelihood of twinning based on the results of step b). In certain embodiments, the estimating step b) comprises a laboratory analysis and step c) comprises a statistical calculation. In other embodiments, step b) comprises a field test, and in specific embodiments may comprises a threshold estimate or a visual indicator of acceptability.

Preferably, the method utilizes at least one sequence that is quantitatively associated with twinning with statistical significance of at least p≦0.01, more preferably, p≦0.005, even more preferably, p≦0.001.

In various embodiments, the method is useful for estimating breeding value in cattle In certain embodiments, each polynucleotide represents one allele of a SNP in the bovine genome.

The method utilizes at least one of the polynucleotides complementary to a sequence located on bovine chromosome 5 (BTA5). More specifically, at least one of the polynucleotides is complementary to a sequence that maps between 55-75 Mb of BTA5; or, more particularly, between 64-73 Mb of BTA5. In another embodiment, one or more of the polynucleotides is complementary to a sequence that maps on BTA5 at any of 24-26 Mb, 49-51 Mb, 64 to 65 Mb, or 69-70 Mb, or, more particularly, between 69.2 to 69.9 Mb.

In another embodiment, the method utilizes at least one polynucleotides complementary to a sequence located in a genomic sequence for Insulin-like Growth Factor 1 (IGF1). Alternatively, at least one of the polynucleotides is complementary to a sequence in a genomic sequence for synapsin III (SYN3), synapsin III isoform A (SYN3A), collagen type X alpha-1 (COL10A 1) precursor, eukaryotic translation initiation factor 3, subunit 7 (EIF3S7), insulin-like growth factor 1 (IGF1), inhibin beta-C (INHBC), cytoskeleton-associated protein 4 (CKAP4), or Alpha24-HS glycoprotein (AHSG.)

In various embodiments, the method utilizes at least one polynucleotide useful for detecting one or more particular SNPs of the bovine genome or BTA5, as described in detail herein.

Another aspect of the invention features a kit for estimating breeding value or predicting the likelihood of twinning comprising: (1) a collection of polynucleotides as described above; and (2) instructions for using the collections for estimating breeding value or predicting the likelihood or genetic potential for twinning.

Other features, and advantages of the present invention will be readily apparent to those skilled in the art, by reference to the drawings, detailed description and examples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Genome-wide single marker association tests. Log likelihood ratio test (LRT) of each marker association test is plotted by SNP in sequential order position (not proportional to Mb location). SNP results are organized by chromosome with chromosome identification indicated along the x-axis. Significant (p=2.3×10⁻⁵. solid line) and suggestive (p=7×10⁻⁴. dotted line) thresholds are indicated with horizontal lines.

FIG. 2: Genome-wide scan using linkage combined with linkage disequilibrium. Log likelihood ratio of linkage only (dark dots) and LLD (light dots) are plotted by the center of haplotype window in sequential order by chromosome (x axis, not proportional to Mb location), Significant (p=2.3×10⁻⁵, solid line) and suggestive (p=7×10⁻⁴, dotted line) thresholds are indicated with horizontal lines.

FIG. 3, Distribution of six-marker windows by physical size. The distribution is shown for distances spanned by six-marker windows across the genome. Bin size was 500 kb.

FIG. 4. Diagram describing phenotypic datasets and genotypic data generated during the progression of the study described in Example 2, which expands on Example 1. Multiple datasets were investigated as data were acquired (Data I, II, III). Cross-validation of two versions of Data III, which depended on how missing genotypes were treated, uncovered a list of SNPs for use in reduced marker panels. Ultimately, two prediction equations (M, NM), reflecting the two versions of Data III, were examined for their efficacy in predicting genetic merit for twinning rate in two versions of Data I.

FIG. 5. Distribution of six-marker windows by physical size. Distances covered by each 6-marker haplotype during LLD analysis are binned according to size. Bin size is 500 kb.

FIG. 6. LLD and association results plotted by physical location. Markers from thirteen regions were analyzed with a LLD (L) and an association (A) analysis utilizing Data I (I) and Data II (II). Regions are labeled according to chromosome and analysis. The −Log₁₀ P-value is plotted according to the relative location of the marker or marker interval in Mb. Solid and dashed horizontal lines indicate −Log₁₀ P-values of 2 and 3, respectively. For the plot of association results (−A), open circles reflect previously genotyped markers from the original significant haplotype (Example 1). Closed circles reflect markers that were added for this analysis. The closed square reflects a marker from the original haplotype that was also found significant in Example 1, and was therefore a marker slated for validation.

FIG. 7. Association results of markers tested for validation of Example 1 results, as well as previously ungenotyped markers. The −Log₁₀ P-value from Data I (I) and Data II (II) are plotted according to chromosome and Mb location (Mb location not to scale). Chromosome UNKN refers to unmapped marker loci. Solid and dashed horizontal lines indicate −Log₁₀, P-values of 2 and 3, respectively. Data points indicated as a filled circle reflect markers not previously genotyped. Data points indicated as a square were previously genotyped. Data points indicated as a solid square in Data II reflect markers now considered validated. They had been found significant in Example 1.

FIG. 8: Linkage disequilibrium (LD) between marker pairs between 5 Mb and 85 Mb on BTA5. LD (D′) and linkage map distance between all marker pairs are plotted. Darkest square indicates high LD (D′>0.8) and lightest square indicates low LD (D′<0.2). Genomic location (Mb) of SNP markers is shown at the top of the LD plot.

FIG. 9. Linkage and combined linkage-linkage disequilibrium (LLD) on chromosome 5. Likelihood ratio test (LRT) of linkage (light solid line) and LLD (dark dotted line) is plotted against center of eight-marker windows. (A) Results obtained using Data I; (B) Results obtained using Data II; (C) Results obtained from both Data 1 and Data II, plotted using multivariate analysis. Thresholds for significant (p<2.3×10⁻⁵, solid line) and suggestive (p<7.2×10⁻⁴, dotted line) linkage levels are indicated.

FIG. 10: Single marker association tests plotted against physical position of SNP on BTA5. (A) Results obtained using Data I; (B) Results obtained using Data II;

FIG. 11. Haplotype block structure of the IGF1 gene. (A) The approximate physical location of each SNP is shown at the top of the figure, and pairwise LD (D′) values are indicated in the triangular figure below. Increasing LD is indicated with darker fill. Two haplotype blocks were observed within the IGF1 locus. (B) The frequency of haplotypes within each block are shown together with the multiallelic D′ between the two blocks (0.54).

GCTCTACTCG; (SEQ ID NO: 371) ACCCCGTTAA; (SEQ ID NO: 372) GCCCTGTTAG; (SEQ ID NO: 373) ATCCCGTAAA; (SEQ ID NO: 374) GCCCTGTTAA; (SEQ ID NO: 375) ACCCTGTTAA; (SEQ ID NO: 376) ATCTCGTAAA. (SEQ ID NO: 377)

DETAILED DESCRIPTION Definitions

The following abbreviations may be used herein:

-   -   cM, centiMorgan;     -   CWER, comparison-wise error rates;     -   FDR; false discovery rate;     -   HWE, Hardy-Weinberg equilibrium;     -   IBD, identity by descent;     -   Kb, kilobase;     -   LD, linkage disequilibrium;     -   LLD, linkage-linkage disequilibrium;     -   LRT, log-likelihood ratio;     -   MAF, minor allele frequency;     -   Mb, megabase;     -   NCBI, National Center for Biotechnology Information;     -   PEV, prediction error variance;     -   PTA, predicted transmitting ability;     -   QTL, quantitative trait loci; and     -   SNP, single nucleotide polymorphism.

The term “individual” when referring to an animal means an individual animal of any species or kind.

The term “animal” is used in a general sense and means a human or other animal, including avian, bovine, canine, equine, feline, hicrine, lupine, murine, ovine, and porcine animals. Preferably the animal is a mammal, particularly a bovine. Unless otherwise specified, or clear from the context, the term “mammal” herein includes human. The term “non-human” animal may be used herein to refer to any animal other than human.

As used herein, “linkage disequilibrium” (or “LD”) refers to allelic association between specific alleles at two or more neighboring loci in the genome, e.g., within a population. LD can be determined for a single marker or locus, or multiple markers. LD is sometimes expressed herein as r² values where r²=1/(4N_(e)c+1) where c=recombination rate (M), and Ne=effective population size. (Sved, 1971)

As used herein, “allele” refers to one or more alternative forms of a particular sequence that contains a SNP. The sequence may or may not be within a gene, and may be within a coding or noncoding portion of such a gene, and may be within an exon or an intron of a particular gene.

“Quantitative trait locus,” (or “QTL”), as used herein is a genomic sequence that is associated with a particular phenotypic trait. Multiple QTL may be identified for a particular trait, and they are frequently found on different chromosomes. The number of QTLs that associate significantly with a particular phenotypic trait may provide an indication of the genetic architecture of a trait, the number of genes that effect the trait, or the extent of the effect of one or more of those genes. One or more QTL that significantly associate with a trait may be candidate genes underlying that trait, which can be sequenced and identified. The significance of the degree of association of a given QTL with a particular trait can be assessed statistically, e.g. through QTL mapping of the alleles that occur in a locus and the phenotypes that they produce. Statistical analysis is preferred to demonstrate whether an observed association with a trait is significant. The presence of a QTL, and its location identify a particular region of the genome as potentially containing a gene that is associated, directly (e.g., structurally) or indirectly (e.g., regulatory) with the trait being analyzed. The probability of association can be plotted for various markers associated with the trait spaced across a chromosome, or throughout the genome.

Positions on chromosomes of the bovine (Bos taurus) genome (BTA) are calculated with reference to bovine genome assembly 3.1 unless otherwise noted.

A “polynucleotide” includes single-stranded or a multi-stranded nucleic acid molecules comprising two or more sequential bases, including any single strand or parallel and anti-parallel strands of a multi-stranded nucleic acid. Polynucleotide may be of any length, and thus, include very large nucleic acids, as well as short ones, such as oligonucleotides.

The term “oligonucleotide” typically refers to short polynucleotides, generally no greater than about 50 nucleotides. It will be understood that if a nucleotide sequence is denoted by a DNA sequence (i.e., A, T, G, C), the corresponding RNA sequence (i.e., A, U, G, C, wherein “U” replaces “T”) is also included.

As used throughout, ranges herein are stated in shorthand, so as to avoid having to set out at length and describe each and every value within the range. Any appropriate value within the range can be selected, where appropriate, as the upper value, lower value, or the terminus of the range. For example, a range of 0.1-1.0 represents the terminal values of 0.1 and 1.0, as well as the intermediate values of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and all intermediate ranges encompassed within 0.1-1.0, such as 0.2-0.5, 0.2-0.8, 0.7-1.0, and so on.

As used herein and in the appended claims, the singular form of a word includes the plural, and vice versa, unless the context clearly dictates otherwise. Thus, the references “a”, “an”, and “the” are generally inclusive of the plurals of the respective terms. For example, reference to “a SNP”, “a method”, or “a trait” includes a plurality of such “SNPs”, “methods”, or “traits.” Reference herein, for example to “an association” includes a plurality of such associations, whereas reference to “chromosomes” includes a single chromosome where such interpretation is not precluded from the context. Similarly, the words “comprise”, “comprises”, and “comprising” are to be interpreted inclusively rather than exclusively. Likewise the terms “include”, “including” and “or” should all be construed to be inclusive, unless such a construction is clearly prohibited from the context. Where used herein the term “examples,” particularly when followed by a listing of terms is merely exemplary and illustrative, and should not be deemed to be exclusive or comprehensive.

The methods and compositions and other advances disclosed here are not limited to particular methodology, protocols, and reagents described herein because, as the skilled artisan will appreciate, they may vary. Further, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to, and does not, limit the scope of that which is disclosed or claimed.

Unless defined otherwise, all technical and scientific terms, terms of art, and acronyms used herein have the meanings commonly understood by one of ordinary skill in the art in the field(s) of the invention, or in the field(s) where the term is used. Although any compositions, methods, articles of manufacture, or other means or materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred compositions, methods, articles of manufacture, or other means or materials are described herein.

All patents, patent applications, publications, technical and/or scholarly articles, and other references cited or referred to herein are in their entirety incorporated herein by reference to the extent allowed by law. The discussion of those references is intended merely to summarize the assertions made therein. No admission is made that any such patents, patent applications, publications or references, or any portion thereof, are relevant, material, or prior art. The right to challenge the accuracy and pertinence of any assertion of such patents, patent applications, publications, and other references as relevant, material, or prior art is specifically reserved. Full citations for publications not cited fully within the specification are set forth at the end of the specification.

Details

The present invention springs in part from the inventors' development of a particularly advantageous approach to identifying genomic regions or genes associated with twinning rate in cattle. The approach first combines the use of linkage and linkage disequilibrium, as well as single-marker association testing using a moderate density whole-genome marker map for twinning rate QTL, to identify putative QTL regions. Twenty independent regions on 14 chromosomes were identified showing significant association for twinning rate (P<0.0007). In addition, 82 and 13 positive single-marker associations reached suggestive and significant thresholds (Lander & Kruglyak, 1995), respectively. Results obtained by this initial approach are then confirmed by (1) validating putative QTL from the linkage-linkage disequilibrium (LLD) analysis results, (2) validating single marker association analyses, and (3) identifying a reduced marker panel to predict genetic merit for twinning rate.

In addition, the inventors have refined the mapping of twinning-rate QTL on bovine chromosome 5 (BTA5) and have identified several positional candidate genes associated with twinning or ovulation rate. Twinning and ovulation rate QTL were mapped previously in the 60-70 Mb genomic region of chromosome 5 (Lien et al. 2000, Cruickshank et al. 2004). Meuwissen et al. (2002) reported the marker bracket of CSSM22 (60.26 Mb) and ILSTS66 (60.27 Mb) showed the highest significance level for twinning rate QTL with the linkage-linkage disequilibrium method. Their result putatively narrowed the genomic region of QTL location to within a 0.7 cM interval between 72.4 cM and 72.9 cM, relative to the USDA-MARC linkage map. However, a significant association between CSSM22 and ILSTS66 and twinning rate may have been detected due to linkage disequilibrium between this marker bracket and a twinning rate QTL outside the region narrowly bounded by these markers. The present inventors have discovered more than two QTL on chromosome 5 through the use of a linkage-linkage disequilibrium analysis with a higher density of SNP markers compared with previous twinning or ovulation rate QTL studies.

Several possible functional candidate genes have also been identified. Of these, IGF1 is a functional candidate gene in the 60-70 Mb QTL region. Association between twinning rate and IGF1 polymorphisms has been previously investigated (Lien et al. 2000, Meuwissen et al. 2002); however, no significant associations between SNPs and twinning rate were identified in that study. In accordance with an embodiment of the present invention, a single marker association testing and linkage-linkage disequilibrium analysis provide support for IGF1 as a candidate gene. As discussed in detail below and in Example 3, IGF1 SNPs 2 and 19 were consistently associated with twinning rate across data sets.

Thus, one aspect of the invention features a collection of polynucleotide sequences, or polynucleotides, each of which is at least partially complementary to a sequence in the bovine genome. The presence or absence of the at least partially complementary sequences, i.e. the sequences in the bovine genome, is quantitatively associated with the trait of twinning in a cattle population. It is known in the art that twinning and ovulation rate are related. Therefore, for purposes herein, it is to be understood that a quantitative association with ovulation rate can be considered a quantitative association with twinning. In various embodiments, the collection comprises at least one sequence that is quantitatively associated with twinning with statistical significance of at least p≦0.01. Preferred are those collections comprising at least one sequence that is quantitatively associated with twinning with statistical significance of at least p≦0.001, or even less. In various embodiments, the statistical significance of the quantitative association with twinning is p≦0.001, p≦0.0009, p≦0.0008, p≦0.0007, p≦0.0006, p≦0.0005, or even less. Most preferred are embodiments that have statistical significance of p≦10⁻⁴, 10⁻⁵, or even 10⁻⁶, or lower. Thus, the more highly significant (i.e., the lower the p value) the association is, the more useful the polynucleotide collection can be for predicting twinning. In certain embodiments, polynucleotides useful for indicating the presence or absence of genomic sequences whose association with twinning is, from a statistical view, only suggestive, may be useful herein. More preferred are those polynucleotides useful for indicating the presence or absence of genomic sequences whose association with twinning is highly suggestive, significant, or even highly significant. The skilled artisan will understand that the statistical significance levels deemed suggestive, highly suggestive, significant, or highly significant will vary based on the particular statistical measures used, and the underlying data used to generate the measures of association. Examples of such statistical measures are shown in the working examples.

The collection of polynucleotides is useful for predicting twinning rate or likelihood of twinning within an individual member of a population, or within a herd, and is also useful for other purposes such as estimating breeding value in cattle, whether for genetic purposes (e.g. breed improvement, herd management, and the like), or for economic considerations (e.g., determining or estimating sale or replacement value of an animal or reproductive material from an animal, predicting the value of offspring, estimating gain or loss for milk or meat production (e.g., practical cost or impact of twinning for farmer), or the like, or a combination thereof.

The polynucleotides in the collection can be any sequences, for example, they could encompass a portion of structural genes, regulatory genes, or other sequences, e.g., SNPs, microsatellite sequences, or other sequences of any length found in a genome. The polynucleotides of the collections may correspond to either strand of a nucleic acid heteroduplex. In some embodiments, the polynucleotides are completely complementary to a portion of a genome, while in others they may be less than completely complementary, provided that they are useful for detecting at least a partially complementary sequence in the genome. For example, in various applications the polynucleotides may be used as primers for amplifying specific sequences to be detected, which may not require 100% complementarity. In other embodiments, the polynucleotide may be used as probes for binding to various sequences to be detected. In one embodiment, each polynucleotide is useful for detecting the presence or absence of one allele of a SNP in the bovine genome. In other embodiments, each polynucleotide comprises one allele of a SNP in the bovine genome, or its complement.

The collection can comprise as few as a single polynucleotide, identifying or comprising one location on the bovine genuine that predicts likelihood of ovulation or twinning. Or the collection can comprise two or more polynucleotides identifying or comprising a region or location on the bovine genome associated with likelihood of ovulation or twinning. Likewise, the collection can comprise two or more polynucleotides identifying or comprising two or more locations or regions in the bovine genome associated with likelihood of ovulation or twinning. By “two or more”, is meant 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 82, 84, 85, 86, 87, 88, 89, 90, 90, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more polynucleotides and/or locations or regions in the bovine genome associated with ovulation or twinning rate. Even larger collections may be useful, including collections comprising 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or more (or incremental numbers in between) polynucleotides and/or locations or regions in the bovine genome associated with ovulation or twinning rate.

The collection can comprise sequences distributed throughout the genome. In one embodiment of the collection, at least one of the polynucleotides is complementary to a sequence located on any bovine chromosome. In one embodiment, the preferred chromosomes include one or more of chromosomes 2, 5, 7, 8, 9, 10, 12, 14, 23, and 26. In another, bovine chromosome 5 (BTA5) is preferred. Especially preferred are particular regions of chromosome 5, including those that are near or encode certain genes. In one embodiment, at least one of the polynucleotides is complementary to a sequence that maps between 55-75 Mb of BTA5. In another embodiment, at least one of the polynucleotides is complementary to a sequence that maps between 64-73 Mb of BTA5.

In various embodiments, the collection comprises one or more polynucleotides complementary to or comprising a sequence that maps on BTA5 at any of 25-35 Mb, 49-51 Mb, 64 to 65 Mb, 64-70 Mb, or 69-70 Mb. Collections comprising polynucleotides complementary to or comprising a sequence that maps on BTA5 between 69.2 to 69.9 Mb are also useful herein. Also useful are polynucleotides that can identify the presence or absence of sequences which map to various overlapping or more specific locations, as set forth in the Examples that follow.

In one embodiment, the collection comprises at least one polynucleotide complementary to a sequence located in a genomic sequence for Insulin-like Growth Factor 1 (IGF1). As mentioned above, IGF1 has been identified herein as a positional candidate that is significantly associated with twinning. Thus, certain preferred collections of polynucleotides feature one or more sequences that can be used to identify the presence or absence of, for example, SNPs within IGF1.

Other genes and sequences near genes may be associated with twinning based on the results. In one embodiment, the collection comprises at least one polynucleotide complementary to a sequence in a genomic sequence for any of synapsin III (SYN3), synapsin III isoform A (SYN3A), collagen type X alpha-1 (COL10A1) precursor, eukaryotic translation initiation factor 3, subunit 7 (EIF3S7), insulin-like growth factor 1 (IGF1), inhibin beta-C (INHBC), inhibin beta-E (INHBE), cytoskeleton-associated protein 4 (CKAP4), small nuclear ribonucleoprotein polypeptide F (SNRPF), or Alpha2-HS glycoprotein (AHSG).

The collection can also comprise at least one polynucleotide useful for detecting one or more specific SNPs. For example, as described in Example 1, the following SNPs have been quantitatively associated with twinning, and are thus sequences for detecting their presence are useful herein: NCBI ID Nos.: ss38323359, ss38323363, ss38323365, ss38323360, ss38323358, ss38323366, ss38325118, ss38323361, ss38329163, ss38324813, ss38331774, ss38323369, ss38336298, ss38335633, ss38326907, ss38336726, ss38323652, ss46526720, ss38330411, ss38332219, ss38325193, ss38335345, ss38333314, ss46526291, ss38330877, ss38336723, ss38335309, ss38333309, ss38333310, ss38333663, ss38329960, ss38330391, ss38322517, ss38323940, ss38334298, ss38332526, ss38336725, ss38328717, ss38322429, ss38331771, ss38327056, ss38336504, ss38323808, ss38335213, ss38324980, ss38330878, ss46526879, ss38331316, ss38331238, ss38328850, ss38330358, ss38322515, ss38333671, ss38328261, ss38331718, ss38324897, ss38336695, ss38331244, ss38322430, ss38328727, ss38326485, ss38324963, ss38324144, ss38332685, ss38324979, ss38324438, ss38327684, ss38329891, ss38328514, ss38328176, ss38325374, ss38328732, ss38325794, ss38336045, ss38326801, ss38323430, ss38327854, ss38334807, ss38331602, ss46526524, ss46527005, ss38329662, ss46526960, ss38324252, ss38331831, ss38327198, ss38328592, ss38322778, ss38332613, ss38330112, or ss38322851, or Affymetrix ID Nos: 351958, 346215, 352891, or 348800.

Other embodiments feature collections which comprise at least one polynucleotide useful for detecting one or more of the SNPs: rs29011599, rs29024681, rs29021155, rs29018125, NGS_(—)38815, IGF1_SNP₂, IBISS4snp1087, BTA_(—)146334, rs29021023, rs41643915, IGF1_SNP_(—)3, rs29020900, 342895, 346579, 353083, 348806, 342533, 348984, 346294, 350040, 353778, 347457, 352924, or 344647.

In various embodiments of the collections or the methods below, the SNPs comprise one or more of the SNPs listed in Table A, which have been associated with likelihood of twinning with the probability of p<0.01.

TABLE A SNPs Useful for Predicting Twinning location location SNP ID Chromosome (Mb)* (bp) Name Rs29011599 BTA23 39.167513 39167513 rs29011599 342895 BTA07 63.145607 rs29011411 BTB_01387869 BTA14 35.836 35836440 BTB-01387869 BTA_34389 BTA14 26.825 26824533 Hapmap44230-BTA-34389 346579 Unassigned 0.1 rs29021107 NGS_25065 BTA14 18.877 18876538 ARS-BFGL-NGS-25065 NGS_102351 BTA14 22.604 22603704 ARS-BFGL-NGS-102351 353083 BTA01 32.137635 rs29009758 NGS_28234 BTA14 19.362 19361957 ARS-BFGL-NGS-28234 348806 BTA23 45.827007 rs29022783 Rs29024681 BTA06 79.523 rs29024681 Rs43711280 BTA06 13.334137 13334137 rs43711280 BTB_00553442 BTA14 15.935 15934673 BTB-00553442 353661 BTA06 79.522733 rs29024685 349058 BTA14 21.397893 rs29010470 Rs29010471 BTA14 19.174 rs29010471 BTC_060018 BTA14 11.023 11022828 Hapmap33448-BTC-060018 Rs29021155 BTA11 60.655 rs29021155 BTB_00566358 BTA14 37.459 37458775 BTB-00566358 UA_IFASA_662 BTA14 10.876 10875942 UA_IFASA_6621 Rs29018125 BTA08 63 65595479 Hapmap57239-rs29018125 345125 BTA07 57.368766 rs29024709 BTB_01640837 BTA14 36.095 36095274 BTB-01640837 Rs43593025 BTA09 36.222498 36222498 rs43593025 345659 BTA23 40.390349 rs29020409 NGS_105600 BTA14 11.985 11985274 ARS-BFGL-NGS-105600 UA_IFASA_974 BTA14 16.788 16787645 UA_IFASA_9744 NGS_38815 BTA14 32.377 32376943 ARS-BFGL-NGS-38815 Rs42576017 BTA09 84.852484 84852484 rs42576017 BTB_00554463 BTA14 16.523 16523349 BTB-00554463 344087 BTA08 58.589588 rs29020823 BTA_35994 BTA14 16.137 16136944 Hapmap41517-BTA-35994 Rs41257131 BTA05 69.956 rs41257131 NGS_74296 BTA14 18.45 18450040 ARS-BFGL-NGS-74296 Rs29015331 BTA12 53.142923 53142923 rs29015331 350925 BTA11 61.111228 rs29026881 351455 BTA01 26.481443 rs29010972 IGF1_SNP_2 BTA05 IGF1_SNP_2 UA_IFASA_535 BTA14 8.133 110348989 UA-IFASA-5357 NGS_32892 BTA14 27.557 27556509 ARS-BFGL-NGS-32892 BTC_064278 BTA14 10.293 10293409 Hapmap23620-BTC-064278 343553 BTA15 15.912146 rs29015662 BTC_009441 BTA14 9.498 9498495 Hapmap27677-BTC-009441 342533 BTA24 56.263923 rs29015105 NGS_68879 BTA14 20.217 20216880 ARS-BFGL-NGS-68879 348984 BTA07 69.93493 rs29012592 Rs29022922 BTA03 99.70654 99706540 rs29022922 351863 BTA06 79.685587 rs29018255 Rs29011598 BTA23 39.161007 39161007 rs29011598 BTA_34285 BTA14 23.32 23320027 Hapmap41234-BTA-34285 Rs29020454 BTA14 39.152227 39152227 rs29020454 344240 BTA15 15.914696 rs29015664 BAC_17596 BTA14 33.736 33735947 ARS_BFGL_BAC_17596 BTC_069412 BTA14 10.768 10768340 Hapmap23304-BTC-069412 UA_IFASA_853 BTA14 12.61 12609632 UA_IFASA_8537 IBISS4snp108 BTA14 IBISS4snp1087 Rs29027275 BTA07 60.306 rs29027275 UA_IFASA_806 BTA14 36.932 36931827 UA_IFASA_8067 345940 BTA01 104.426701 rs29016850 346294 BTA14 35.942595 rs29012557 Rs29012205 BTA03 99.650394 111990685 Hapmap60034-rs29012205 BTB_00564573 BTA14 34.474 34473718 BTB-00564573 Rs29014046 BTA14 26.072 26072216 Hapmap60213-rs29014046 BTA_42171 BTA14 15.515 15515273 Hapmap38314-BTA-42171 BTA_146334 BTA09 35.45476 35454760 Hapmap27597-BTA-146334 350040 BTA06 41.880315 rs29015348 350255 BTA12 43.324066 rs29025358 BTB_00983321 BTA14 36.845 36844856 BTB-00983321 UA_IFASA_817 BTA14 38.245 38245445 UA_IFASA_8173 Rs29018356 BTA26 5.419816 5071319 Hapmap60861-rs29018356 353778 BTA04 33.77989 rs29010454 Rs41257060 BTA18 9.180167 9180167 rs41257060 Rs29023153 BTA18 81.93884 5720430 Hapmap54020-rs29023153 NGS_109705 BTA26 8.343595 8343595 ARS-BFGL-NGS-109705 BTC_058037 BTA14 8.377 8376618 Hapmap22733-BTC-058037 351958 BTA14 35.63073 N/A BTA_117246 BTA14 20.421 20420773 Hapmap50246-BTA-117246 351128 BTA04 44.351856 rs29017408 Rs41630079 BTA23 22.458431 22458431 rs41630079 ARS_USMARC_2 BTA14 14.615 14614797 ARS_USMARC_20 347929 BTA13 13.907594 rs29023434 Rs41654960 BTA06 13.076723 13076723 rs41654960 NGS_39645 BTA22 41.805633 41805633 ARS-BFGL-NGS-39645 343419 Unassigned 0.1 rs29019730 352477 BTA13 13.90762 rs29023435 BTC_065402 BTA14 25.007 25006867 Hapmap23524-BTC-065402 NGS_117252 BTA14 9.99 9990358 ARS-BFGL-NGS-117252 Rs41257406 BTA11 95.986 rs41257406 Rs41622614 BTA14 39.251444 39251444 rs41622614 346113 Unassigned 0.1 rs29022856 350005 BTA02 24.13038 rs29014793 Rs29021023 BTA26 35.766 rs29021023 Rs29021895 BTA14 11.53 rs29021895 348422 BTA18 11.239122 rs29021176 NGS_112623 BTA14 18.683 18683218 ARS-BFGL-NGS-112623 347656 BTA15 5.131999 rs29026137 348966 BTA01 136.701531 rs29012088 347181 BTA01 3.098043 rs29012841 345586 BTA07 60.772232 rs29014932 BTC_010226 BTA14 11.748 11748067 Hapmap23784-BTC-010226 347457 BTA28 7.107731 rs29020955 Rs29011274 BTA01 83.619259 83619259 rs29011274 Rs29010308 BTA05 16.836 17272102 Hapmap55237-rs29010308 352924 BTA06 20.51922 rs29024705 352831 BTA14 11.826138 rs29025879 Rs43596422 BTA09 35.670291 35670291 rs43596422 Rs41643915 BTA22 45.735107 45735107 rs41643915 345483 BTA11 43.717906 rs29010503 343311 BTA11 43.71812 rs29010502 BTB_00555233 BTA14 18.078 18078367 BTB-00555233 IGF1_SNP_3 BTA05 IGF1_SNP_3 344647 BTA25 13.02804 rs29010042 Rs29020019 BTA14 21.191 21191021 Hapmap60530-rs29020019 Rs29020900 BTA14 19.91 19910198 Hapmap54398-rs29020900 349178 BTA08 53.33558 rs29015209 BTC_057468 BTA14 8.85 8850017 Hapmap30158-BTC-057468 347863 BTA05 53.606067 rs29018610 IGFBP5_SNP_1 BTA02 IGFBP5_SNP_1 BTC_009295 BTA14 9.265 9264515 Hapmap31246-BTC-009295 354320 BTA20 45.25198 rs29017434 NGS_37911 BTA14 14.274 14274020 ARS-BFGL-NGS-37911 351968 BTA01 24.821808 rs29013556 345460 BTA25 12.919628 rs29010040 NGS_36089 BTA14 23.89 23890322 ARS-BFGL-NGS-36089 Rs29013534 BTA26 41.299 rs29013534 354046 BTA10 86.478816 rs29025325 342709 Unassigned 0.1 rs41256874 Rs29014603 BTA24 37.894 rs29014603 Rs43151146 BTA12 53.520216 53520216 rs43151146 BTB_01417924 BTA14 22.383 22382726 BTB-01417924 IGF1_SNP_4 BTA05 IGF1_SNP_4 NGS_95360 BTA23 21.816693 21816693 ARS-BFGL-NGS-95360 352758 BTA04 39.4991 rs29027100 NGS_104239 BTA14 12.861 12860774 ARS-BFGL-NGS-104239 IGFBP2_INDEL BTA02 Rs29015262 BTA01 83.898922 83898922 rs29015262 BTA_75794 BTA14 17.342 17342324 BTA-75794-no-rs 353440 BTA06 13.627253 rs29013621 BTA_160247 BTA14 32.005 32004992 Hapmap33516-BTA-160247 IGFBP2_SNP_3 BTA02 IGFBP2_SNP_3 Rs29010821 BTA20 23.299301 23299301 rs29010821 343530 BTA06 15.070137 rs29025372 352263 BTA05 58.47649 rs29021931 Rs29013606 BTA09 29.112806 29112806 rs29013606 IGF1_SNP_5 BTA05 IGF1_SNP_5 Rs29010828 BTA20 23.295081 23295081 rs29010828 NGS_117284 BTA14 18.537 18537116 ARS-BFGL-NGS-117284 Rs41617651 BTA20 23.756215 23756215 rs41617651 345993 BTA11 46.665435 rs29024171 IGF1_SNP_19 BTA05 IGF1_SNP_19 351743 BTA08 49.45456 rs29016114 BTC_073113 BTA14 25.332 25332174 Hapmap23509-BTC-073113 *(Mb location refers to Bovine Genome Assembly 3.1)

Many SNPs have been logged in various databases and contain identification numbers specific to those databases. Table B shows a correlation between ID numbers for various SNPs useful in the practice of the present invention.

TABLE B Affymetrix Affymetrix ID Baylor ID NCBI rs ID ID Baylor ID NCBI rs ID 347181 BTA-01887 rs29012841 351977 BTA-00615 rs29013769 351968 BTA-00399 rs29013556 350255 BTA-09192 rs29025358 351455 BTA-03206 rs29010972 353615 BTA-09639 rs29025797 353083 BTA-02200 rs29009758 347929 BTA-14673 rs29023434 345940 BTA-11086 rs29016850 352477 BTA-14674 rs29023435 343232 BTA-00073 rs29013238 343395 BTA-05004 rs29019171 343414 BTA-05466 rs29019625 349503 BTA-10446 rs29015999 348966 BTA-01121 rs29012088 352831 BTA-09722 rs29025879 352894 BTA-11634 rs29017393 349058 BTA-02927 rs29010470 350005 BTA-04433 rs29014793 351644 BTA-08306 rs29021868 346734 BTA-09626 rs29025784 351958 IBISS4snp1088 N/A 345489 BTA-03092 rs29010859 346294 BTA-01600 rs29012557 344064 BTA-05801 rs29019957 342962 BTA-06677 rs29021035 349032 BTA-02521 rs29010075 347656 BTA-09981 rs29026137 352758 BTA-07435 rs29027100 346278 BTA-01166 rs29012133 351128 BTA-11650 rs29017408 348104 BTA-01167 rs29012134 344152 BTA-08060 rs29021627 343553 BTA-10096 rs29015662 350968 BTA-08059 rs29021626 344240 BTA-10098 rs29015664 343051 BTA-11379 rs29017141 349746 IBISS4snp306 N/A 352891 BTA-11485 rs29017245 348422 BTA-06819 rs29021176 350649 BTA-00799 rs29013950 351009 BTA-08826 rs29022382 352263 BTA-08373 rs29021931 346682 BTA-08825 rs29022381 353440 BTA-00465 rs29013621 354320 BTA-11676 rs29017434 343530 BTA-09206 rs29025372 342875 BTA-02845 rs29010390 352924 BTA-12755 rs29024705 352801 BTA-08873 rs29022429 350040 BTA-04998 rs29015348 345659 BTA-06043 rs29020409 352302 BTA-09550 rs29025709 348806 BTA-14016 rs29022783 353661 BTA-12733 rs29024685 342533 BTA-04749 rs29015105 351863 BTA-13293 rs29018255 345460 BTA-02486 rs29010040 345125 BTA-12759 rs29024709 344647 BTA-02488 rs29010042 345586 BTA-04573 rs29014932 349617 BTA-12782 rs29024732 342895 BTA-03650 rs29011411 345949 BTA-11257 rs29017020 348984 BTA-01635 rs29012592 353888 BTA-11258 rs29017021 351743 BTA-10561 rs29016114 343540 BTA-09719 rs29025876 349178 BTA-04855 rs29015209 347457 BTA-06596 rs29020955 344087 BTA-06462 rs29020823 343092 BTA-13993 rs29018944 354046 BTA-09159 rs29025325 350252 BTA-09108 rs29025275 350212 BTA-08458 rs29022016 344205 BTA-09264 rs29025430 347686 BTA-10474 rs29016027 344809 BTA-06209 rs29020573 345483 BTA-02962 rs29010503 346579 BTA-06750 rs29021107 343311 BTA-02961 rs29010502 343419 BTA-05571 rs29019730 345993 BTA-12216 rs29024171 349771 IBISS4snp767 rs41257204 352905 BTA-12213 rs29024168 342709 IBISS4snp425 rs41256874 350925 BTA-07215 rs29026881 352215 BTA-06885 rs29021241 350642 BTA-00616 rs29013770 343032 BTA-10318 rs29015876

In one embodiment, the following table (Table C) can be used to construct a polynomial equation for predicting the association of a particular SNP or collection of SNPs with the trait of twinning, as described in Example 1.

TABLE C Factors for predicting twinning using specific SNPs SNP ID Coefficient Std. Error P-value Gene (Intercept) −0.052452 0.050677 0.300962 — rs29011599 0.027694 0.006733 4.30e−05 — rs29024681 0.049162 0.007790 4.53e−10 — rs29021155 0.022143 0.006070 0.000281 — rs29018125 −0.025313 0.005904 2.02e−05 — NGS_38815 −0.050368 0.006401 1.13e−14 — IGF1_SNP_2 0.041562 0.006320 8.58e−11 IGF1 IBISS4snp1087 −0.058557 0.007438 1.09e−14 AHSG BTA_146334 0.040993 0.005929 9.46e−12 — rs29021023 −0.023879 0.007995 0.002903 — rs41643915 −0.019959 0.006234 0.001418 — IGF1_SNP_3 0.043182 0.007457 9.97e−09 — rs29020900 0.024738 0.007881 0.001757 — 342895 −0.036595 0.006397 1.48e−08 — 346579 −0.021105 0.004676 7.30e−06 — 353083 −0.023775 0.005242 6.61e−06 — 348806 −0.053445 0.006548 1.23e−15 — 342533 0.040633 0.012344 0.001038 — 348984 0.022913 0.006718 0.000679 — 346294 −0.027682 0.007883 0.000469 — 350040 −0.031963 0.006259 4.08e−07 — 353778 0.030813 0.006404 1.78e−06 — 347457 0.049421 0.006018 8.34e−16 — 352924 −0.044905 0.016661 0.007176 — 344647 −0.030730 0.009300 0.000993 —

In a preferred embodiment, SNPs that have been further validated as described in Example 2 are particularly useful to identify with polynucleotides of the invention. These include, from Table 2-2: rs29012841, rs29013556, rs29010972, rs29009758, rs29016850, rs29012088, rs29014793, rs29010454, rs29017408, rs29010308, rs29018610, IGF1_SNP_(—)3, IGF1_SNP_(—)2, rs41257131, rs29024681, rs29024705, rs29015348, rs29018255, rs29024685, rs29024709, rs29014932, rs29027275, rs29011411, rs2901259, rs29015209, rs29020823, rs29018125, BTA_(—)146334, rs29019730, rs29025325, rs29010503, rs29010502, rs29021155, rs29026881, rs41257406, rs29025358, rs29023435, rs29023434, rs29021895, rs29025879, NGS_(—)74296, NGS_(—)25065, rs29010471, NGS_(—)28234, rs29020900, rs29010470, rs29014046, NGS_(—)70865, NGS_(—)38815, BAC_(—)17596, BTB_(—)005645, IBISS4snp1087, AffyID3519, rs29012557, BTB_(—)013878, rs41622614, rs29015662, rs29021176, rs29022856, rs29020409, rs29022783, rs29015105, rs29010042, rs29010040, rs29015664, rs29021023, and rs29020955.

Following backward elimination analysis as described in Example 2, a reduced marker panel was developed (see Table 2-3), containing the following 18 SNPs: rs29010454, IGF1_SNP_(—)2, IGF1_SNP_(—)3, rs29015348, rs29024681, rs29012592, rs29011411, rs29020823, rs29015209, BTA_(—)146334, rs29010503, AffyID3519, IBISS4snp1087, BTB_(—)005645, NGS_(—)28234, rs29015662, rs29022783 and rs29020955. This information and the other information set forth in Example 2 and Table 2-3 in particular may be used to predict an animal's genetic merit for twinning rate. For example, an animal can be genotyped for up to all 18 of the SNPs listed above, then using the coefficients of Table 2-3 corresponding to its set of genotypes, the values would be totaled to arrive at the animal's predicted twinning rate.

Still other SNPs that are useful in connection herewith include various SNPs on BTA5, particularly SNPs within the IGF1 region, or within the introns of IGF1 specifically, especially intron 2 of IGF1. For example SNP2, 5 and 19 have been found to be highly and consistently associated with twinning. In particular, the inventors have discovered that an allelelic substitution of G to A in IGF SNP2 was associated with a reduction of twinning rate. The reduction was consistent across two independent data sets. Surprisingly, despite suspicions of associations, prior studies looked for, but found no significant associations between IGF1 SNPs and twinning rate. Several SNPs associated with twinning within the IGF1 gene are set forth in Table 3-5.

In addition, SNPs on bovine chromosome 5 (BTA5) associated with likelihood of twinning (see Table 3-4) include: ss38332405, ss38334594, ss38324419, ss38324418, ss38333537, ss38324813, ss46526664, 54966387, 54966493, 54977448, ss38322168, 58655362, 63551646, 63554840, 64273448, 65538715, ss46527005, ss46526677 and ss38322778.

It will be understood by the skilled artisan that the SNPs or other polymorphisms identified herein as markers of ovulation or twinning rate are associated with genomic regions associated with these traits. As such, preferred embodiments of the methods of the invention include identifying any of the SNPs exemplified herein, as well as any SNPs or polymorphisms within the same genomic region. In one embodiment, that genomic region comprises +/−10,000 bases of the identified SNP or other polymorphism. In another embodiment, the genomic region comprises +/−20,000 bases of the identified SNP or other polymorphism. In another embodiment, the genomic region comprises +/−30,000 bases of the identified SNP or other polymorphism. In another embodiment, the genomic region comprises +/−40,000 bases of the identified SNP or other polymorphism. In another embodiment, the genomic region comprises +/−50,000 bases of the identified SNP or other polymorphism. In another embodiment, the genomic region comprises +/−60,000 bases of the identified SNP or other polymorphism. In another embodiment, the genomic region comprises +/−70,000 bases of the identified SNP or other polymorphism. In another embodiment, the genomic region comprises +/−80,000 bases of the identified SNP or other polymorphism. In another embodiment, the genomic region comprises +/−90,000 bases of the identified SNP or other polymorphism. In another embodiment, the genomic region comprises +/−100,000 bases of the identified SNP or other polymorphism. In another embodiment, the genomic region comprises +/−110,000 bases of the identified SNP or other polymorphism. In another embodiment, the genomic region comprises +/−120,000 bases of the identified SNP or other polymorphism. In another embodiment, the genomic region comprises +/−130,000 bases of the identified SNP or other polymorphism. In another embodiment, the genomic region comprises +/−140,000 bases of the identified SNP or other polymorphism. In another embodiment, the genomic region comprises +/−150,000 bases of the identified SNP or other polymorphism. In another embodiment, the genomic region comprises +/−160,000 bases of the identified SNP or other polymorphism. In another embodiment, the genomic region comprises +/−170,000 bases of the identified SNP or other polymorphism. In another embodiment, the genomic region comprises +/−180,000 bases of the identified SNP or other polymorphism. In another embodiment, the genomic region comprises +/−190,000 bases of the identified SNP or other polymorphism. In another embodiment, the genomic region comprises 200,000 bases of the identified SNP or other polymorphism.

In another of its several aspects, the invention features methods of detecting sequences in a genome that provide an estimate of twinning probability or which have predictive value regarding twinning likelihood. In one embodiment, methods for estimating the likelihood of twinning in one or more members of a cattle population are provided. The methods generally comprise the steps of:

a) providing a collection of one or more polynucleotides, each of which is at least partially complementary to a sequence in a bovine genome, comprising or identifying at least one sequence that is quantitatively associated with twinning with statistical significance of at least p≦0.01;

b) using the collection to determine the presence or absence of sequences complementary to one or more polynucleotides from the collection in one or more members of the cattle population genome, wherein the presence or absence of the complementary sequences is quantitatively associated with the trait of twinning in a cattle population; and

c) estimating the likelihood of twinning based on the results of step b).

The method, as the skilled artisan will appreciate, encompass use of collections of polynucleotides, for example, as described above, which are useful for detecting the presence or absence of sequences in a genome that are predictive of twinning. In one embodiment, the estimating step comprises a laboratory analysis. In such embodiments, the method comprises a statistical calculation. In other embodiments, the method comprises a field test. In many such embodiments, preferred tests are conveniently used to provide a threshold estimate or a visual indicator of acceptability. Preferably, no actual statistical calculation is required for such field tests. Such tests may require the use of a chart, or reader other device to provide a measurement of twinning rate, or other useful measurement or result that reflects the likelihood of twinning.

Preferably, the methods provided herein feature a collection of polynucleotides that comprises at least one sequence that is quantitatively associated with twinning with statistical significance of at least p≦0.01. In other embodiments, the collection comprises at least one sequence that is quantitatively associated with twinning with statistical significance of at least p≦0.005. Most preferred are methods wherein the collection comprises at least one sequence that is quantitatively associated with twinning with statistical significance of at least p≦0.001.

The method may involve the use of a collection comprising as few as a single polynucleotide, identifying one location on the bovine genome that predicts likelihood of ovulation or twinning. Or the method may utilize two or more polynucleotides identifying a region or location on the bovine genome associated with likelihood of ovulation or twinning. Likewise, the method may utilize a collection comprising two or more polynucleotides identifying two or more locations or regions in the bovine genome associated with likelihood of ovulation or twinning. By “two or more”, is meant 2, 3, 4, 5, 6, 7, 8, 9, 10, II, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 82, 84, 85, 86, 87, 88, 89, 90, 90, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more polynucleotides and/or locations or regions in the bovine genome associated with likelihood of ovulation or twinning. Even larger collections may be used, including collections comprising 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or more (or incremental numbers in between) polynucleotides and/or locations or regions in the bovine genome associated with ovulation or twinning rate.

The methods preferably are useful for estimating breeding value in cattle, thus preferably feature a collection of polynucleotides that is useful for estimating breeding value in cattle.

In various embodiments, the collection is useful for detecting the presence or absence of one allele of a SNP in the bovine genome. Preferably, at least one of the polynucleotides in the collection is complementary to a sequence located on bovine chromosome 5 (BTA5).

In certain embodiments of the methods, at least one of the polynucleotides in the collection is complementary to a sequence that maps between 55-75 Mb of BTA5, between 64-73 Mb of BTA5, or at any of 25-35 Mb, 49-51 Mb, 64 to 65 Mb, or 69-70 Mb of BTA5.

In a particular method, one or more of the polynucleotides in the collection is complementary to a sequence that maps on BTA5 between 69.2 to 69.9 Mb. Even more preferred are methods wherein at least one of the polynucleotides in the collection is complementary to a sequence located in a genomic sequence for Insulin-like Growth Factor 1 (IGF1).

In various embodiments, the method features collections of polynucleotides wherein at least one of the polynucleotides in the collection is complementary to a sequence in a genomic sequence for synapsin III (SYN3), synapsin III isoform A (SYN3A), collagen type X alpha-1 (COL10A1) precursor, eukaryotic translation initiation factor 3, subunit 7 (EIF3S7), insulin-like growth factor 1 (IGF1), inhibin beta-C (INHBC), inhibin beta-E (INHBE), cytoskeleton-associated protein 4 (CKAP4), small nuclear ribonucleoprotein polypeptide F (SNRPF), or Alpha2-HS glycoprotein (AHSG).

The method can also utilize a collection comprising at least one polynucleotide useful for detecting one or more specific SNPs. For example, as described in Example 1, the following SNPs have been quantitatively associated with twinning, and are thus sequences for detecting their presence are useful herein: NCBI ID Nos.: ss38323359, ss38323363, ss38323365, ss38323360, ss38323358, ss38323366, ss38325118, ss38323361, ss38329163, ss38324813, ss38331774, ss38323369, ss38336298, ss38335633, ss38326907, ss38336726, ss38323652, ss46526720, ss38330411, ss38332219, ss38325193, ss38335345, ss38333314, ss46526291, ss38330877, ss38336723, ss38335309, ss38333309, ss38333310, ss38333663, ss38329960, ss38330391, ss38322517, ss38323940, ss38334298, ss38332526, ss38336725, ss38328717, ss38322429, ss38331771, ss38327056, ss38336504, ss38323808, ss38335213, ss38324980, ss38330878, ss46526879, ss38331316, ss38331238, ss38328850, ss38330358, ss38322515, ss38333671, ss38328261, ss38331718, ss38324897, ss38336695, ss38331244, ss38322430, ss38328727, ss38326485, ss38324963, ss38324144, ss38332685, ss38324979, ss38324438, ss38327684, ss38329891, ss38328514, ss38328176, ss38325374, ss38328732, ss38325794, ss38336045, ss38326801, ss38323430, ss38327854, ss38334807, ss38331602, ss46526524, ss46527005, ss38329662, ss46526960, ss38324252, ss38331831, ss38327198, ss38328592, ss38322778, ss38332613, ss38330112, or ss38322851, or Affymetrix ID Nos: 351958, 346215, 352891, or 348800.

Other embodiments of the method utilize collections which comprise at least one polynucleotide useful for detecting one or more of the SNPs: rs29011599, rs29024681, rs29021155, rs29018125, NGS_(—)38815, IGF1_SNP_(—)2, IBISS4snp1087, BTA_(—)146334, rs29021023, rs416-43915, rs29020900, 342895, 346579, 353083, 348806, 342533, 348984, 346294, 350040, 353778, 347457, 352924, or 344647.

In one embodiment of the method, the information in Table C above can be used to construct a polynomial equation for predicting the association of a particular SNP or collection of SNPs with the trait of twinning, as described in Example 1.

In a preferred embodiment, the method is used to query the bovine genome for SNPs that have been further validated as described in Example 2. These include, from Table 2-2: rs29012841, rs29013556, rs29010972, rs29009758, rs29016850, rs29012088, rs29014793, rs29010454, rs29017408, rs29010308, rs29018610, IGF1_SNP3, IGF1_SNP_(—)2, rs41257131, rs29024681, rs29024705, rs29015348, rs29018255, rs29024685, rs29024709, rs29014932, rs29027275, rs29011411, rs2901259, rs29015209, rs29020823, rs29018125, BTA_(—)146334, rs29019730, rs29025325, rs29010503, rs29010502, rs29021155, rs29026881, rs41257406, rs29025358, rs29023435, rs29023434, rs29021895, rs29025879, NGS_(—)74296, NGS_(—)25065, rs29010471, NGS_(—)28234, rs29020900, rs29010470, rs29014046, NGS_(—)70865, NGS_(—)38815, BAC_(—)17596, BTB_(—)005645, IBISS4snp1087, AffyID3519, rs29012557, BTB_(—)013878, rs41622614, rs29015662, rs29021176, rs29022856, rs29020409, rs29022783, rs29015105, rs29010042, rs29010040, rs29015664, rs29021023, and rs29020955.

Following backward elimination analysis as described in Example 2, a reduced marker panel has been developed (see Table 2-3), containing the following 18 SNPs: rs29010454, IGF1_SNP_(—)2, IGF1_SNP_(—)3, rs29015348, rs29024681, rs29012592, rs29011411, rs29020823, rs29015209, BTA_(—)146334, rs29010503, AffyID3519, IBISS4snp1087, BTB_(—)005645, NGS_(—)28234, rs29015662, rs29022783 and rs29020955. This information and the other information set forth in Example 2 and Table 2-3 in particular, may be used to predict an animal's genetic merit for twinning rate. For example, an animal can be genotyped for up to all 18 of the SNPs listed above, then using the coefficients of Table 2-3 corresponding to its set of genotypes, the values are totaled to arrive at the animal's predicted twinning rate.

Still other SNPs that are useful in these methods include various SNPs on BTA5, particularly SNPs within the IGF1 region, or within the introns of IGF1 specifically, especially intron 2 of IGF1. For example SNP2, 5 and 19 have been found to be highly and consistently associated with twinning. In particular, the inventors have discovered that an allelelic substitution of G to A in IGF SNP2 was associated with a reduction of twinning rate. The reduction was consistent across two independent data sets.

In addition, SNPs on bovine chromosome 5 (BTA5) associated with likelihood of twinning (see Table 3-4) include: ss38332405, ss38334594, ss38324419, ss38324418, ss38333537, ss38324813, ss46526664, 54966387, 54966493, 54977448, ss38322168, 58655362, 63551646, 63554840, 64273448, 65538715, ss46527005, ss46526677 and ss38322778.

As mentioned above, preferred embodiments of the method of the invention involves genotyping one or more animals to detect the presence of one or more polymorphisms that can be quantitatively associated with twinning rate, or transmission of twinning rate, in the animals. In particular, the polymorphisms comprise SNPs. An exemplary embodiment utilizes the reduced marker panel and attendant information shown in Table 2-3, and the prediction equation(s) described in Example 2.

Genotyping for polymorphisms, including SNPs, is well known in the art. Numerous methods and technology platforms are commercially available to accomplish SNP genotyping, and the skilled artisan will understand which methods are most suited to the purposes described herein. Methods particularly suitable to genome-wide and more localized genotyping of animals are set forth herein in Examples 1, 2 and 3.

It will be understood by the skilled artisan that the SNPs or other polymorphisms identified herein as markers of ovulation or twinning rate are associated with genomic regions associated with these traits. As such, preferred embodiments of the methods of the invention include identifying any of the SNPs exemplified herein, as well as any SNPs or polymorphisms within the same genomic region. In one embodiment, that genomic region comprises +/−10,000 bases of the identified SNP or other polymorphism. In another embodiment, the genomic region comprises +/−20,000 bases of the identified SNP or other polymorphism. In another embodiment, the genomic region comprises +/−30,000 bases of the identified SNP or other polymorphism. In another embodiment, the genomic region comprises +/−40,000 bases of the identified SNP or other polymorphism. In another embodiment, the genomic region comprises +/−50,000 bases of the identified SNP or other polymorphism. In another embodiment, the genomic region comprises +/−60,000 bases of the identified SNP or other polymorphism. In another embodiment, the genomic region comprises +/−70,000 bases of the identified SNP or other polymorphism. In another embodiment, the genomic region comprises +/−80,000 bases of the identified SNP or other polymorphism. In another embodiment, the genomic region comprises +/−90,000 bases of the identified SNP or other polymorphism. In another embodiment, the genomic region comprises +/−100,000 bases of the identified SNP or other polymorphism. In another embodiment, the genomic region comprises +/−110,000 bases of the identified SNP or other polymorphism. In another embodiment, the genomic region comprises +/−120,000 bases of the identified SNP or other polymorphism. In another embodiment, the genomic region comprises +/−130,000 bases of the identified SNP or other polymorphism. In another embodiment, the genomic region comprises +/−140,000 bases of the identified. SNP or other polymorphism. In another embodiment, the genomic region comprises +/−150,000 bases of the identified. SNP or other polymorphism. In another embodiment, the genomic region comprises +/−160,000 bases of the identified SNP or other polymorphism. In another embodiment, the genomic region comprises +/−170,000 bases of the identified SNP or other polymorphism. In another embodiment, the genomic region comprises +/−180,000 bases of the identified SNP or other polymorphism. In another embodiment, the genomic region comprises +/−190,000 bases of the identified SNP or other polymorphism. In another embodiment, the genomic region comprises +/−200,000 bases of the identified SNP or other polymorphism.

In yet another of its several aspects, the invention features kits that comprise one or more of the collections of polynucleotides useful for detecting sequences in a genome that are quantitatively associated with twinning, and instructions for use of the collection(s) for estimating breeding value or predicting the likelihood of twinning.

The kits can comprise as few as a single polynucleotide, identifying one location on the bovine genome that predicts likelihood of ovulation or twinning. Or the kits can comprise two or more polynucleotides identifying a region or location on the bovine genome associated with likelihood of ovulation or twinning. Likewise, the kits can comprise two or more polynucleotides identifying two or more locations or regions in the bovine genome associated with ovulation or twinning rate. By “two or more”, is meant 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 82, 84, 85, 86, 87, 88, 89, 90, 90, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more polynucleotides and/or locations or regions in the bovine genome associated with likelihood of ovulation or twinning. Even larger collections may be useful, including collections comprising 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or more (or incremental numbers in between) polynucleotides and/or locations or regions in the bovine genome associated with ovulation or twinning rate.

In one embodiment the kit comprises one or more PCR primer sets for amplifying a genomic region associated with twinning rate. In another embodiment, the kit comprises comprising one or more nucleic acid probe sets that specifically bind to a genomic region comprising a SNP or other feature associated with twinning rate. The kit may further include one or more reagents for the detection or capture of such SNPs or other features, or information pertaining thereto. In one embodiment, the kit comprises a polynucleotide microarray. The kit may also include computer programs or software to perform calculations used in the various methods described herein.

The kits preferably contain polynucleotides useful for estimating breeding value in cattle. In various embodiments, the kits are useful for detecting the presence or absence of one allele of a SNP in the bovine genome. Preferably, at least one of the polynucleotides in the kit is complementary to a sequence located on bovine chromosome 5 (BTA5).

In certain embodiments, at least one of the polynucleotides in the kit is complementary to a sequence that maps between 55-75 Mb of BTA5, between 64-73 Mb of BTA5, or at any of 25-35 Mb, 49-51 Mb, 64 to 65 Mb, or 69-70 Mb of BTA5.

In certain embodiments, the kit may contain one or more polynucleotides complementary to a sequence that maps on BTA5 between 69.2 to 69.9 Mb. Even more preferred are kits in which at least one of the polynucleotides is complementary to a sequence located in a genomic sequence for Insulin-like Growth Factor 1 (IGF1).

In various embodiments, the kits comprise polynucleotides wherein at least one is complementary to a sequence in a genomic sequence for synapsin III (SYN3), synapsin III isoform A (SYN3A), collagen type X alpha-1 (COL10A1) precursor, eukaryotic translation initiation factor 3, subunit 7 (EIF3S7), insulin-like growth factor 1 (IGF1), inhibin beta-C (INHBC), inhibin beta-E (INHBE), cytoskeleton-associated protein 4 (CKAP4), small nuclear ribonucleoprotein polypeptide F (SNRPF), or Alpha2-HS glycoprotein (AHSG).

The kits can also comprise at least one polynucleotide useful for detecting one or more specific SNPs. For example, as described in Example 1, the following SNPs have been quantitatively associated with twinning, and are thus sequences for detecting their presence are useful herein: NCBI ID Nos.: ss38323359, ss38323363, ss38323365, ss38323360, ss38323358, ss38323366, ss38325118, ss38323361, ss38329163, ss38324813, ss38331774, ss38323369, ss38336298, ss38335633, ss38326907, ss38336726, ss38323652, ss46526720, ss38330411, ss38332219, ss38325193, ss38335345, ss38333314, ss46526291, ss38330877, ss38336723, ss38335309, ss38333309, ss38333310, ss38333663, ss38329960, ss38330391, ss38322517, ss38323940, ss38334298, ss38332526, ss38336725, ss38328717, ss38322429, ss38331771, ss38327056, ss38336504, ss38323808, ss38335213, ss38324980, ss38330878, ss46526879, ss38331316, ss38331238, ss38328850, ss38330358, ss38322515, ss38333671, ss38328261, ss38331718, ss38324897, ss38336695, ss38331244, ss38322430, ss38328727, ss38326485, ss38324963, ss38324144, ss38332685, ss38324979, ss38324438, ss38327684, ss38329891, ss38328514, ss38328176, ss38325374, ss38328732, ss38325794, ss38336045, ss38326801, ss38323430, ss38327854, ss38334807, ss38331602, ss46526524, ss46527005, ss38329662, ss46526960, ss38324252, ss38331831, ss38327198, ss38328592, ss38322778, ss38332613, ss38330112, or ss38322851, or Affymetrix ID Nos: 351958, 346215, 352891, or 348800.

Other embodiments feature kits comprising at least one polynucleotide useful for detecting one or more of the SNPs: rs29011599, rs29024681, rs29021155, rs29018125, NGS_(—)38815, IGF1_SNP_(—)2, IBISS4snp1087, BTA_(—)146334, rs29021023, rs416-43915, rs29020900, 342895, 346579, 353083, 348806, 342533, 348984, 346294, 350040, 353778, 347457, 352924, or 344647.

In one embodiment, the information in Table C above can be used to construct a polynomial equation for predicting the association of a particular SNP or collection of SNPs with the trait of twinning, as described in Example 1. Kits comprising polynucleotides comprising or identifying such SNPs may be assembled.

In a preferred embodiment, the kits are used in a method to query the bovine genome for SNPs that have been further validated as described in Example 2. These include, from Table 2-2: rs29012841, rs29013556, rs29010972, rs29009758, rs29016850, rs29012088, rs29014793, rs29010454, rs29017408, rs29010308, rs29018610, IGF1_SNP_(—)3, IGH_SNP_(—)2, rs41257131, rs29024681, rs29024705, rs29015348, rs29018255, rs29024685, rs29024709, rs29014932, rs29027275, rs29011411, rs2901259, rs29015209, rs29020823, rs29018125, BTA_(—)146334, rs29019730, rs29025325, rs29010503, rs29010502, rs29021155, rs29026881, rs41257406, rs29025358, rs29023435, rs29023434, rs29021895, rs29025879, NGS_(—)74296, NGS_(—)25065, rs29010471, NGS_(—)28234, rs29020900, rs29010470, rs29014046, NGS_(—)70865, NGS_(—)38815, BAC_(—)17596, BTB₁₃ 005645, 1BISS4snp1087, AffyID3519, rs29012557, BTB_(—)013878, rs41622614, rs29015662, rs29021176, rs29022856, rs29020409, rs29022783, rs29015105, rs29010042, rs29010040, rs29015664, rs29021023, and rs29020955.

Following backward elimination analysis as described in Example 2, a reduced marker panel has been developed (see Table 2-3), containing the following 18 SNPs: rs29010454, IGF1_SNP_(—)2, IGF1_SNP_(—)3, rs29015348, rs29024681, rs29012592, rs29011411, rs29020823, rs29015209, BTA_(—)146334, rs29010503, AffyID3519, IBISS4snp1087, BTB_(—)005645, NGS_(—)28234, rs29015662, rs29022783 and rs29020955. This information and the other information set forth in Example 2 and Table 2-3 in particular, may be used to predict an animal's genetic merit for twinning rate. For example, an animal can be genotyped for up to all 18 of the SNPs listed above, then using the coefficients of Table 2-3 corresponding to its set of genotypes, the values are totaled to arrive at the animal's predicted twinning rate. Kits comprising polynucleotides and other reagents useful for identifying these genomic regions may be constructed.

Still other SNPs that are useful for constructing kits include various SNPs on BTA5, particularly SNPs within the IGF1 region, or within the introns of IGF1 specifically, especially intron 2 of IGF1. For example SNP2, 5 and 19 have been found to be highly and consistently associated with twinning. In particular, the inventors have discovered that an allelelic substitution of G to A in IGF SNP2 was associated with a reduction of twinning rate. The reduction was consistent across two independent data sets.

In addition, SNPs on bovine chromosome 5 (BTA5) associated with likelihood of twinning (see Table 3-4) include: ss38332405, ss38334594, ss38324419, ss38324418, ss38333537, ss38324813, ss46526664, 54966387, 54966493, 54977448, ss38322168, 58655362, 63551646, 63554840, 64273448, 65538715, ss46527005, ss46526677 and ss38322778.

EXAMPLES

The invention can be further illustrated by the following examples, although it will be understood that these examples included merely for purposes of illustrating and better describing certain aspects of what is disclosed herein. The examples do not limit the scope of the invention unless otherwise specifically indicated.

Example 1

Single marker association with twinning rate was tested across the entire genome in the North American Holstein population. The results of the single marker association tests were also compared with the results of QTL analysis from linkage mapping and linkage disequilibrium mapping.

The number of SNPs required for a genome-wide scan using association tests and linkage combined with linkage disequilibrium (LLD) were estimated based on the linkage disequilibrium pattern observed in the North American Holstein population.

Materials and Methods:

All animals used in this study were registered Holstein bulls from the United States and Canada. The sons used were from 19 paternal-half sib families and were chosen for having twinning rate predicted transmitting ability (PTA, Johanson et al. 2001) of high accuracy and values in the upper and lower tails for the distribution within family if there were more than approximately 3.0 sons in a family. Mean and median number of daughter records per sire, for calculation of PTAs, were 1071 and 513, respectively. In addition, sons were selected for balanced representation of maternal grandsires within family. Three extended families consisted of two, four, and seven half-sib families, extending to two, three, and four generations, respectively. The other families were one generation half-sib families.

DNA was extracted from semen samples using a modified phenol-chloroform extraction protocol as previously described (Cruickshank et al. 2004). The Affymetrix Bovine 10k SNP panel (Affymetrix. CA) was used to genotype 233 bulls. Thirty-three of the 233 failed in genotyping. Of the remaining samples, sixty-eight were provided as whole-genome amplified DNA using the GenomiPhi kit (Amersham Biosciences, Piscataway, N.J.) and all others were provided as genomic DNA. A total of 9,910 SNP markers obtained from the bovine genome sequencing project were typed. These SNPs were selected from across the genome considering genomic distribution and minor allele frequency (MAF). Eight percent of selected SNPs existed within exons and 60% were located within 50 kb upstream or downstream of genes. Genotypes were tested for Hardy-Weinberg equilibrium to identify possible typing errors.

Marker mar construction: Linkage analysis was performed using CRIMAP 2.4 (Green et al. 2004). Physical locations of SNP markers were available from genomic assembly v3.1. If the location of a marker based on linkage mapping was different (>30 cM) from its expected position, based on physical genomic information, it was considered as a genome assembly error or a genotyping error. Those SNPs were excluded for association tests and LLD analysis. Double recombinations between closely-linked loci (<5 cM) were considered as genotyping errors. This type of genotyping error detected as departures from Mendelian inheritance were treated as missing data.

Haplotype construction: Haplotypes were constructed using information on partial haplotypes inherited from sires, as well as linkage distance between closely-linked markers. This method extended minimization of recombination to include all informative markers in offspring (Windig and Meuwissen, 2004). The most probable paternally inherited haplotype of offspring in each half-sib family was obtained using the chrompic option of CRIMAP. Paternal haplotype of each half-sib was inferred using paternally-inherited alleles and linkage phase in offspring inferred by CRIMAP. After the paternal allele was identified, the other allele was deduced as the maternally-inherited allele in offspring. This procedure was implemented using a per1 script.

Genome-wide Association Tests: To reduce false-positive errors, 6,154 markers along 8,483 physically-mapped SNPs were selected for testing twinning rate association, based on having a genotype call rate greater than 80% and a minor allele frequency greater than 5%. Association between a marker locus and twinning rate PTA was evaluated with a model including effects of sire and SNP genotype. A weighted least square analysis was performed using the known error variance structure obtained from the PTA prediction error variance (PEV).

Statistical thresholds for a genome-wide search were determined, accounting for multiple comparisons. To decide the threshold level for a genome-wide scan, an analytical method proposed by Lander and Kruglyak (1995) was used. The genome-wide significance level corresponding to exceeding threshold (T) is calculated using μ(T)=[C+2ρGT²]α(T), where C is the number of chromosomes, ρ is the expected rate of recombination per Morgan, G is the genome length in Morgans, and α(T) is the point-wise significance level of exceeding level T. For LLD and association tests, is two, considering within-family and historical recombinations. C is 30 and G is 30. Comparison-wise error rates (CWER) of 2.3×10⁻⁵ and 7.2×10⁻⁴ yielded experiment-wise error rates corresponding to the significant and suggestive levels. For linkage QTL threshold, significant (p=5.0×10⁻⁵) and suggestive level (p=0.002) were calculated with ρ=1.

Linkage disequilibrium between markers was measured to evaluate genome coverage of single marker association tests using the Affymetrix bovine 10K SNP panel (Affymetrix, CA). LD was calculated with all pairs of SNPs within a 10 Mb genomic region of the same chromosome. Pairwise r²>0.5 between SNPs within 500 kb on autosomal chromosomes was considered as the threshold for estimating the genomic region captured by tag SNPs, which were representative of adjacent SNPs. SNPs selected for genome-wide association tests were used to estimate r². Linkage disequilibrium was analyzed using Haploview software (Barrett et al. 2004).

Linkage and Linkage Disequilibrium QTL Mapping: The LLD analysis estimated QTL effects associated with paternal and maternal alleles after accounting for background genetic effects on twinning rate. Identity by descent (IBD) within family was estimated based on haplotype relationship within family and identity by descent probabilities of non-informative genomic regions between sire and offspring was estimated using informative flanking markers. IBD probabilities were calculated using marker haplotypes and the coalescent theory (Meuwissen and Goddard, 2001). IBD probabilities were calculated using a window of four or six markers. If a six-marker window extended greater than three Mb, the window was reduced to four markers to minimize errors in the IBD prediction by spurious LD. Polygenic linkage and linkage disequilibrium effects were estimated as random effects in the following linear model:

y=Zu+Wq+e

where: y is a vector of twinning rate PTAs; W is an incidence matrix of haplotypes relating phenotypic records to QTL alleles; Z is a diagonal incidence matrix relating individual polygenic effects to individual sons; u and q are random effects assumed to be distributed u˜(0, Aσ_(u) ²) and q˜(0, Gσ_(q) ²), where σ_(u) ², and σ_(q) ² are the polygenic variance and the additive QTL variance, respectively; A is the matrix explaining additive genetic relationship among animals; and G is the allele relationship (IBD) matrix between haplotypes.

Polygenic and random QTL effects were estimated using the ASReml package (Gilmour et al. 2002). The log likelihood of the data under models of linkage only or LLD was compared with that under the null hypothesis of no QTL effect at the central marker bracket of the four- or six-marker windows. Analyses were performed for all marker windows across the genome, advancing the window marker-by-marker.

Simulation to Evaluate Predictive Accuracy of Model: A simulation was used to evaluate accuracy of identity by descent prediction between two haplotypes in a population representing that used here, i.e., corresponding SNP density and linkage disequilibrium pattern. A population of 1,000 animals obtained from random selection and mating of 100 males and 100 females (N_(e)=200) was simulated for 300 generations. Simulation conditions were chosen based on producing an LD pattern corresponding to that observed here for the North American dairy cattle population. A window size of 0.2-2.0 Mb consisting of a biallelic QTL locus located in the center of six evenly-spaced SNP markers was simulated to estimate IBD probability of the biallelic locus at the middle of the six-marker window. All loci were set in complete LD in the founder population with the two haplotypes having equal frequencies. In the final population, 100 haplotypes from different individuals were randomly sampled to calculate IBD probability of the QTL. To evaluate accuracy of IBD prediction, an average of the absolute difference between predicted IBD probability and true IBD probability was calculated using 100 replications. Linkage disequilibrium (r²) was measured in the sampled population. Linkage map distance was approximated so that 1 Mb was equivalent to 1 cM. This simulation was implemented using R (www.r-project.org).

Results:

Genotypes for genome-wide scan: The results of the SNP screening selection process are summarized in Table 1-1. Ninety SNP markers that deviated from the Hardy-Weinberg equilibrium (HWE) were considered as genotyping error (p<0.001). Linkage analysis was used to identify SNPs that were positioned incorrectly. Compared to bovine genome assembly 3,1, the genomic position of 295 autosomal SNPs deviated more than 30 Mb from the position inferred by linkage mapping based on the assumption that 1 cM was equivalent to 1 Mb. This was less than 5% of SNPs selected for linkage analysis. The percentage of non-Mendelian inheritance errors in genotypes was less than 0.5% of the total genotypes. Although marker loci were not evenly distributed across the genome, mean and median of interval distance between adjacent loci selected for the genome-wide scan was 0.31 Mb and 022 Mb on the bovine genome assembly 3.1. Total size of the bovine genome assembly 3.1 was approximately 2,400 Mb. Of 7,146 SNPs selected for the genome wide scan, 1,964 SNPs were located within 50 kb upstream or downstream of genes and 2,667 SNPs existed within known genes. The remaining SNPs (2,515) were located in intergenic regions. According to the available genomic annotation of databases at the National Center for Biotechnology Information (NCBI) (url is ncbi.nlm.nih.govsmapview) over 20,000 genes were annotated on the bovine genome assembly 3.1 using transcribed sequences, known sequence from other species, or bioinformatics gene prediction tools.

TABLE 1-1 Summary of SNP markers selection. Selected Eliminated SNP screening method SNPs SNPs Original genotypes 9,919 — Physically mapped in genome assembly 3.1 8,433 1,436 Genotyping success rate > 80% 8,018 465 Hardy-Weinberg equilibrium test (p < 0.001) 7,928 90 Heterozygous in at least one sire (for linkage 7,492 436 analysis) Incorrect locus order or Mendelian 7,146 346 segregation errors (selection for LLD) Minor allele exists in two or more half-sib 6,454 809 families & minor allele frequency > 5% (selection for marker association test)

Coverage evaluation by linkage disequilibrium: Linkage disequilibrium (LD). (r²) was measured between pairs of closely positioned loci across the bovine genome. Linkage disequilibrium was evaluated for 14,140 SNP pairs within 500 kb of each other. Mean and median r² between all SNP pairs within 500 kb were 0.28 and 0.09. The mean genomic distance between marker pairs was 22.5 kb at a threshold of r²>0.5. This distance decreased to 6.5 kb at r²>0.8. On the simplified assumption that a SNP captures the variation of other SNPs associated at r²>0.5, coverage with the 6,545 SNPs used in the association testing was approximately 150-300 Mb of the bovine genome at the r²=0.5 threshold. When r²=0.2 was used as a threshold of LD, the mean distance between two loci was 73.3 kb. LD was weak (r²<0.1) when the genomic distance between two markers was over 100 kb. LD of few marker pairs (<0.5%) extended up to 500 kb at the r²=0.5 threshold.

Single marker-phenotype association test: The genome-wide scan detected potential association between markers and twinning rate on most chromosomes. This test produced 95 positive associations (FIG. 1) at the suggestive threshold (p<0.0007). Of the 95 SNPs associated with twinning rate, thirteen loci were associated at the significant threshold level, p<2.3×10⁻⁵. (see Table 1-2). Significant (p<2.3×10⁻⁵) associations were located on chromosome 2 (nine SNPs), chromosome 14 (three SNPs) and chromosome 5 (one SNP). The most significant marker association accounted for 23% of the variance in twinning rate PTA, corresponding to approximately 2% of the phenotypic variance for twinning rate.

TABLE 1-2 Genome-wide significant and suggestive single marker associations with bovine twinning rate. SNP ID (NCBI dbSNP) Chromosome Position (bp) Significance (p) 351958* 14 35630730 6.1E−08 ss38323359 2 102908665 2.6E−07 ss38323363 2 102908937 2.7E−07 346215* 14 35630276 3.4E−07 ss38323365 2 102909009 3.5E−07 ss38323360 2 102908818 5.8E−07 ss38323358 2 102908604 1.2E−06 ss38323366 2 102909120 2.1E−06 ss38325118 2 60717386 1.0E−05 ss38323361 2 102908874 1.2E−05 ss38329163 2 107664703 1.4E−05 ss38324813 5 16835901 1.7E−05 ss38331774 14 11826138 2.1E−05 ss38323369 2 102909189 3.0E−05 ss38336298 5 74152696 3.2E−05 ss38335633 5 94167125 3.3E−05 ss38326907 8 53335580 3.3E−05 ss38336726 13 13907620 3.5E−05 ss38323652 14 35942595 3.6E−05 ss46526720 16 44201685 3.7E−05 ss38330411 18 44352521 3.7E−05 ss38332219 26 4139063 3.8E−05 ss38325193 16 48530530 4.4E−05 ss38335345 6 79685587 5.2E−05 ss38333314 11 90404790 5.3E−05 ss46526291 15 19270036 7.1E−05 ss38330877 18 24417031 7.7E−05 ss38336723 13 13907157 8.3E−05 ss38335309 20 66751407 8.6E−05 ss38333309 26 25816607 8.7E−05 ss38333310 26 25816776 8.9E−05 ss38333663 10 14728252 9.4E−05 ss38329960 24 59173205 9.8E−05 ss38330391 14 11950458 1.0E−04 ss38322517 6 13627253 1.1E−04 ss38323940 1 3097998 1.1E−04 ss38334298 6 18142242 1.2E−04 ss38332526 11 40666353 1.2E−04 ss38336725 13 13907594 1.3E−04 ss38328717 26 35765651 1.3E−04 ss38322429 26 41299476 1.3E−04 ss38331771 27 12798363 1.3E−04 ss38327056 13 38888532 1.4E−04 ss38336504 5 67908996 1.5E−04 ss38323808 8 22450832 1.5E−04 ss38335213 8 63000355 1.5E−04 ss38324980 14 21397838 1.5E−04 ss38330878 18 24416870 1.5E−04 ss46526879 15 70362925 1.6E−04 ss38331316 28 31402223 1.7E−04 ss38331238 5 10929300 1.8E−04 ss38328850 11 60654721 1.8E−04 ss38330358 14 29438577 1.8E−04 ss38322515 7 55292134 1.9E−04 ss38333671 13 6391027 2.0E−04 ss38328261 28 37620053 2.1E−04 ss38331718 14 37472909 2.2E−04 ss38324897 21 41141313 2.4E−04 ss38336695 22 48871417 2.5E−04 ss38331244 12 43324066 2.6E−04 ss38322430 26 41304563 2.6E−04 ss38328727 5 17309229 2.8E−04 ss38326485 2 24130380 2.9E−04 ss38324963 4 33779890 3.0E−04 ss38324144 9 28629530 3.1E−04 ss38332685 27 20207785 3.3E−04 ss38324979 14 21397893 3.5E−04 ss38324438 10 61109327 3.8E−04 ss38327684 5 62463316 3.9E−04 ss38329891 27 21184304 3.9E−04 ss38328514 8 58589588 4.1E−04 ss38328176 20 52237178 4.1E−04 ss38325374 4 52832040 4.2E−04 ss38328732 8 99165959 4.2E−04 352891* 5 12826659 4.3E−04 ss38325794 11 40729983 4.3E−04 ss38336045 28 29997007 4.3E−04 ss38326801 24 56263923 4.4E−04 ss38323430 5 117885058 4.7E−04 ss38327854 3 95595378 4.9E−04 ss38334807 6 20519220 5.1E−04 348800* 2 70113131 5.2E−04 ss38331602 6 42472402 5.2E−04 ss46526524 1 138435263 5.4E−04 ss46527005 5 69688243 5.4E−04 ss38329662 7 60306431 5.7E−04 ss46526960 11 95986323 5.7E−04 ss38324252 1 32137635 5.8E−04 ss38331831 8 63904909 5.8E−04 ss38327198 11 98893419 5.8E−04 ss38328592 14 22133626 5.8E−04 ss38322778 5 84526473 6.1E−04 ss38332613 8 49454560 6.6E−04 ss38330112 4 105383388 6.8E−04 ss38322851 5 25542789 6.8E−04 *Affymetrix Bovine GeneChip ID.

Linkage combined with linkage disequilibrium mapping: A total of 7,146 loci on autosomal chromosomes used in the linkage and LD scan were chosen for being informative in at least one half-sib family. Average distance between informative markers was approximately 1 Mb or 1 cM in all families. A linkage effect surpassing a suggestive level (p<0.002) was detected only on chromosome 23 (FIG. 2). The LLD scan detected 26 suggestive QTL (p<0.0007) (Table 1-3). These suggestive QTL effects were detected on fifteen chromosomes (FIG. 2). No significant (p<2.3×10⁻⁵) QTL regions were detected across the genome. The most significant QTL was detected on chromosome 9 (p=2.5×10⁻⁵), which was also supported by weak linkage (p<0.01).

TABLE 1.3 Genome-wide suggestive bovine twinning rate QTL using LLD. Center of Six- 3^(rd) and 4^(th) of six-marker marker six-marker window window window (NCBI dbSNP ID) Chromosome (Mb) (Mb) P-value ss38330838-ss38322499 9 29.092 0.856 2.5E−05 ss38322561-ss38337181 12 50.214 2.042* 2.9E−05 ss38325409-ss38336770 12 49.751 1.094 6.3E−05 ss38332854-ss38328543 2 116.349 1.56 6.7E−05 ss38332214-ss46526423 18 47.732 0.962 9.2E−05 ss38325561-ss38325562 1 83.619 2.238* 1.5E−04 ss38323369-ss38327053 2 116.612 2.324 1.8E−04 ss38331300-ss38329161 9 74.021 1.24 1.8E−04 ss38325408-ss38325409 12 49.618 1.278 2.3E−04 ss38323081-ss38335448 26 5.285 2.16 2.7E−04 ss46526082-ss38332663 23 38.623 1.903 2.9E−04 ss38330235-ss46526082 23 38.127 2.156 2.9E−04 ss38322529-ss38322531 6 7.927 0.236 3.0E−04 ss38325164-ss38323757 22 42.684 0.949 3.6E−04 ss38336088-ss38323292 3 99.576 1.412 3.9E−04 ss38330033-ss38330031 10 21.292 0.823 4.1E−04 ss38330766-ss38330767 9 80.493 0.941 4.6E−04 ss38335585-ss38335584 20 23.445 0.351 4.6E−04 ss38336442-ss38325081 18 8.206 0.755* 4.6E−04 ss38336770-ss38322561 12 49.941 2.365 4.7E−04 ss38322389-ss38322388 23 17.256 0.135* 4.7E−04 ss38322510-ss38322328 11 2.615 2.454 5.2E−04 ss38335045-ss38335044 26 3.838 0.807 5.3E−04 ss38335623-ss46526297 20 35.674 0.871 5.6E−04 ss38331067-ss46526233 2 124.49 1.365 6.0E−04 ss38324775-ss46526399 4 94.348 1.608 6.2E−04 *Four-marker window size (six-marker size > 2.5 Mb)

Accuracy of IBD Prediction: The marker density of the genomic regions was considered in deciding numbers of markers (four or six) in each window for estimating IBD. Distribution of six-marker window size is shown in FIG. 3. The mean and median of six-marker windows were 2.58 Mb and 1.42 Mb, respectively. For six-marker windows extending over 0.1 Mb, an average LD between adjacent marker pairs within the same bracket was r²<0.1.

Table 1-4 shows the relationship between accuracy of IBD estimation and LD using simulated data. The simulation was performed with four different marker spacings with six markers located within 0.2, 0.5, 1.0, or 2.0 cM. The number of generations used was 300. The marker spacing and number of generations were selected to obtain a LD pattern similar to the typical breakage of LD observed using six-marker brackets across the bovine genome in this study. The mean error of IBD prediction using 6 marker windows, calculated as mean absolute value of the difference between true (IBD=0 or 1) and predicted identity by decent probability was lower than 0.15, for LD of r²>0.6. As LD decayed to r² in the range of 0.3 to 0.1, the error of IBD prediction increased to 0.25˜0.3 (Table 1-3).

TABLE 1-4 Linkage disequilibrium, linkage map distance and error in IBD prediction. Size of six-marker Mean LD Average deviation from windows (cM/Mb) (r²) (±s.d.)¹ true IBD (±s.d.)² 0.2 0.671 (±0.016) 0.136 (±0.025) 0.5 (0.369 (±0.017)   0.227 (±0.020) 1.0 0.139 (±0.013) 0.319 (±0.017) 2.0 0.021 (±0.004) 0.391 (±0.008) ¹LD between adjacent marker pairs within six-marker windows was used to calculate mean LD (r²). ²An average of the absolute difference between predicted IBD probability and true IBD probability.

Comparison between association tests and LLD: To compare the methods, the genomic regions identified by single marker association tests were compared to QTL identified with LLD analysis. Of 87 SNPs associated with twinning rate at a suggestive level (p<0.0007), only one marker, on chromosome 26, was located within a four- or six-marker windows detected by LLD (p<0.0007).

Example 2

In Example 1, a genome-wide search with a moderate density 10K marker set identified many marker associations with twinning rate either through single marker analysis or combined linkage-linkage disequilibrium (haplotype) analysis. The objective of the study described in this example was to validate putative marker associations using an independent set of phenotypic data.

Materials and Methods:

Experimental outline: A general layout of the study is diagramed in FIG. 4. Briefly, the current study expands on the original set forth in the previous example, but with additional sires being genotyped in the phenotypic dataset (Data I) collected from 1994 to 1998. Genotyping was also performed in an additional dataset comprised of records collected from 1999 to 2006 (Data II). Animals in Data II had minimal overlap with the animals in the original Data I. Single marker and LLD analyses were performed on Data I and Data II. Additional data subsequently became available for sires in Data II, and is referred to as Data III. A cross-validation step, followed by a stepwise backward elimination analysis, was performed on this expanded dataset. The accuracy for which the reduced marker panel was able to predict genetic merit for twinning was analyzed. Marker-predicted breeding values were compared to phenotypic values of twinning in Data I.

Data: Animals used in this study included 921 registered Holstein bulls from 100 paternal half-sib families. Eleven pedigrees consisted of 71 half-sib families extending two or more generations. Twenty-nine pedigrees consisted of single generation pedigrees, and accounted for 11% of the total genotyped individuals. Twinning rate predicted transmitting ability scores (PTAs) were calculated using calving records from 1994 to 1998 (Data I) and 1999 to 2006 (Data II), and the underlying liability scores from threshold model analysis were used as the trait in marker association analyses (Johanson et al. 2001; see Example 1). Data I and Data II consisted of 434 and 851 bulls with twinning rate PTAs, respectively. Three hundred sixty-four bulls were in common between the two datasets. Individuals constituting Data II consisted of 11 pedigrees extending two or more generations, and 20 pedigrees extending one generation. Data I consisted of 9 pedigrees extending two or more generations, and 11 pedigrees extending one generation.

Additional calving records (from 2007-2008) subsequently became available at a later stage of the analysis. New records were added to the original data composing Data II (expanded data referred to herein as Data III). Adjusted daughter averages (Data III) were calculated for genotyped sires and consequently utilized as the phenotype in marker cross-validation analyses. Daughter averages were calculated utilizing daughter calving records adjusted for herd, parity, year, and month of calving.

The calculation of the PTA phenotype is described in detail in Johanson et al. (2001) and in the previous example. Briefly, single or twin births recorded on cows were analyzed in a model including fixed effects of parity (p), herd-year (hy), season (n), and sire group of calf (g), along with random effects of cow's sire(s) and the residual (ε).

y _(ijklmn) =μ+p _(i) +hy _(i) +n _(k) +s _(m)+ε_(ijklmn)

The season effect included 4 levels signifying the quarter of the year in which the calves were born. Unknown sires were grouped into four groups based on birth-year quarter of the calf. From the assessment of twinning rate PTAs, bulls were chosen satisfying requirements of high accuracy of prediction and PTA values in the upper and lower tails of the within-family distribution. In addition, selection accounted for maternal grandsire, intending to balance representation and avoid stratification within the sample population. During all analyses, twinning rate PTAs (Data I, Data II) were weighted by the inverse of their prediction error variance, while adjusted daughter averages (Data III) were weighted by the number of daughters of each sire.

Markers: Markers were selected based on results from Example 1. Markers implicated in significant single-marker associations (P<0.001), as well as those located in significant LLD regions (Table 2-1), were genotyped. Additional previously ungenotyped markers were selected for genotyping. The intension for these added markers was to increase marker density of regions targeted in the LLD analysis. Therefore, relating to their past use with the data presented here, both previously genotyped and previously ungenotyped markers are incorporated in this analysis.

TABLE 2-1 LLD region descriptions. Start Region No. of Position Size Average Median Average BTA SNPs (Mb)⁺ (Mb)⁺ spacing (kb) spacing (kb) r²⁽*⁾  1 9 83.61 5.21 650.66 281.42 0.184  2 10 111.93 1.51 167.54 182.13 0.300  3 9 99.80 4.54 568.10 393.52 0.206  6 10 11.29 1.73 191.72 206.06 0.327  9a 9 79.74 3.09 440.97 278.66 0.287  9b 8 33.62 1.62 202.13 174.98 0.025 12 12 51.89 4.79 435.12 232.48 0.115 18 10 6.26 4.48 498.31 658.74 0.053 20 10 21.82 2.06 228.39 180.24 0.396 22 11 40.13 9.95 995.26 756.98 0.136 23b 9 17.98 7.36 818.24 514.38 0.341 23a 10 39.29 7.06 882.82 883.16 0.075 26 12 4.75 5.34 485.51 168.09 0.179 ⁽*⁾Average correIation coefficient between adjacent marker pairs. ⁺Megabase

DNA was extracted from semen samples using a modified phenol-chloroform extraction protocol as described previously (Cruickshank et al. 2004). Genotyping service was performed using competitive allele-specific PCR or single-base primer extension followed by mass spectrometry detection. Upon completion of marker genotyping, genotype data were subjected to both marker and sample triage. Departure from Hardy Weinberg Equilibrium (HWE) was determined, and markers were excluded from analysis if the observed frequencies were in excess of expectation based on χ² test (P<1×10⁻⁷). Markers having minor allele frequencies (MAF) less than 0.02 were excluded from analysis. Markers having fewer than 80% of the samples successfully genotyped were discarded. Individuals having fewer than 80% of the markers successfully genotyped were also discarded.

Based on the aforementioned criteria, the final list of markers successfully utilized in the analysis encompassed 24 markers genotyped by Sequenom Inc. (San Diego, United States), 104 markers genotyped by DNA Landmarks Inc. (Saint-Jean-sur-Richelieu, Canada), and 178 markers genotyped by Kbiosciences Inc. (Hoddesdon, United Kingdom), for a total of 306 genotyped SNP markers. Physical location of SNP markers were assigned based on the most recent build of the bovine genome assembly (build 4.0). Genome coordinates for markers residing on BTA14 were assigned based on UMD 2.0 assembly (Zimin et al. 2009) due to relocation of markers from the earlier assembly of build 4.0. Linkage disequilibrium (r²) was assessed on all possible combinations of marker pairs within each chromosome segment. The HAPLOVIEW software (Barrett & Cardon 2006) was utilized for this procedure.

Linkage-linkage disequilibrium tests: The presence of QTL was evaluated utilizing a linkage (paternal inheritance) combined with linkage disequilibrium (maternal inheritance) (LLD) analysis. Haplotype information, identity by descent (IBD) probabilities, and analysis of variance modeling contributed to the evidence for or against QTL presence. Haplotypes were constructed from the pedigree and genotype data. Maternal and paternal haplotypes were predicted by simulated annealing using a Markov chain Monte Carlo (MCMC) algorithm within SIMWALK2 (Sobel & Lange 1996). Prior to haplotype determination, testing of Mendelian-inconsistent and Mendelian-consistent inheritance was performed. Individuals having a posterior mistyping probability greater than 0.10 at any locus were treated as missing genotypes for that locus (approximately 1.7% of the genotype data).

As described by Meuwissen & Goddard (2001), calculation of IBD probabilities at marker midpoints was performed. Each calculation utilized the information from a six-marker haplotype window. IBD probabilities were estimated by walking across the genomic regions of interest, interval by interval. Thirteen chromosome regions showing positive results for the presence of QTL in Example 1, were targeted (see Table 2-1 above).

The amount of variance accounted for by the polygenic, paternal and maternal QTL effects was estimated using the following model:

y=Z _(s) u _(s) +Z _(d) u _(d) +W _(p) q _(p) +W _(m) q _(m) +e

where y is a vector of twinning rate PTAs, Z_(s) and Z_(d) are incidence matrices relating individual polygenic effects of sire and dam to individual bulls, W_(p) and W_(m) are incidence matrices relating phenotypic records to paternally and maternally inherited QTL haplotype probabilities. The random effects u_(s), u_(d), q_(p) and q_(m) were assumed to be normally distributed u˜N(0, Aσ² _(u)) and q˜N(0, Gσ² _(q)), where σ² _(u) and σ² _(q) are the sire/dam and additive paternal/maternal QTL variances, respectively. A signifies the paternal/maternal relationship matrices, and G signifies the IBD probability matrix between haplotypes. Polygenic and random QTL effects were estimated using the ASReml package of Gilmour (2002).

The log likelihood of the data under a LLD model with a QTL present was compared to the null hypothesis of a model excluding a QTL effect in linkage disequilibrium. The test statistic took the form of a likelihood ratio test (LRT)

LRT=−2((log_(e)(L _(s))−log_(e)(L _(g)))

where L_(s) is the likelihood of the reduced model and L_(g) is the likelihood of the full model. The LRT follows a χ⁻² _(df=2) distribution under the null hypothesis of no difference between models.

Single-marker association tests: Single-marker associations were analyzed in a weighted least squares regression approach. Twinning rate PTAs were weighted by the inverse of their prediction error variance (PEV) and analyzed for significant association with SNP genotype using a model which included the genotype code (0, 1, or 2 copies of allele I present) and sire effect acquired from pedigree. Statistical threshold levels suitable for a replication study (P<0.01) as proposed by Lander & Kruglyak (1995) were used in this validation.

Cross-validation: As is the case with most statistical software applications, observations are eliminated from a dataset if they have missing data for any fixed effect within the analysis model. This phenomenon occurred while performing the backward elimination analysis with multiple markers. Therefore, missing genotypes were predicted. In the creation of a new version if Data III referred to herein as NM, missing values were reassigned with the most frequent genotype as indicated by the population genotypic frequencies within Data II. The RandomForest package within the statistical software R (URL at R-project.org/) was utilized to create the missing genotype transformation.

A conservative panel of markers for use in the final prediction equation may be chosen through cross-validation. Secondarily, efficacy of prediction equations may be tested on data subsets through cross-validations. To achieve this, Data 111 was broken down into ten data subsets (one-tenth in size to Data III). Subsets were created by random sampling within family without replacement from Data III (i.e. family structure maintained as much as possible within data subsets). Nine data subsets were combined to create a training dataset nine-tenths the original size of Data III for model development. The remaining one-tenth constituted a testing dataset for assessment of model efficacy. This was repeated 10 times, subsequently combining all possible combinations of data subsets into training and testing datasets. A generalized linear model (PROC GLM) was utilized in a stepwise backward elimination approach with SAS statistical analysis software (SAS Institute Inc., Cary, N.C.) on each training dataset with the purpose of identifying a reduced panel of markers for use in predicting genomic breeding values. Markers whose association tests passed validation criterion were available for entry into the model. Markers were allowed to remain in the model providing the significance level remained below threshold (P<0.001).

Prediction Equations: Genotype coefficients were determined for markers remaining in the reduced marker panel from each training set (ie. 1 reduced panel from each of 10 training sets) using a GLM model (SAS Institute Inc., Cary, N.C.). Marker-predicted adjusted daughter averages were calculated in each testing subset utilizing the genotype coefficients derived from the training set. Weighted Pearson correlation coefficients were calculated between true and marker-predicted adjusted daughter averages in each testing subset.

A final reduced marker panel was constructed after stepwise backward elimination analysis of the 10 training datasets. Markers represented at high frequency (i.e., in half or more) were chosen for inclusion in this panel. Marker genotype coefficients were then determined utilizing the entire Data III. These markers and their coefficients formed the final prediction equation which was later applied in Data Ito predict genomic breeding values. Weighted correlations of marker-predicted adjusted daughter averages with true adjusted and true unadjusted daughter averages and PTAs was performed to determine accuracy of the final reduced marker panel. Lastly, markers chosen for the final reduced marker panel were checked for their association with other dairy production traits. The same model as described in the single marker association analyses for twinning rate was used. Available production traits included daughter yield deviations for milk, protein, fat, fat percent, protein percent, and milk protein, as well as daughter deviations for somatic cell score (SCS), productive life (PL), and daughter pregnancy rate (DPR).

Results:

Triage of the genotype data resulted in 13 markers discarded for low MAP (<2%), 7 markers for departure from HWE, and one marker for having fewer successfully genotyped samples than the set threshold. Sixty-two individuals were discarded from all analyses due to having fewer successfully genotyped markers than the set threshold. An additional mistyping analysis was performed as required by SIMWALK2 software (Sobel & Lange 1996; Sobel et al. 2002) prior to haplotype estimation. In total, 2,093 genotypes were treated as missing based on having a posterior mistyping probability greater than 0.10, for a total I.69% mistyping rate.

Genome coordinates of the original markers in each LLD region were based on build 3.1 of the bovine genome. More accurate genomic locations have been assigned during build 4.0, resulting in relocation of a few of the original markers. As a result, the spacing of a few markers at the end of the marker group was larger than desired, and is reflected in the comparison of the median and average marker spacing (Table 2-1).

TABLE 2-1 LLD region descriptions. Start Region No. of Position Size Average Median Average BTA SNPs (Mb)⁺ (Mb)⁺ spacing (kb) spacing (kb) r²⁽*⁾  1 9 83.61 5.21 650.66 281.42 0.184  2 10 111.93 1.5I 167.54 182.13 0.300  3 9 99.80 4.54 568.10 393.52 0.206  6 10 11.29 1.73 191.72 206.06 0.327  9a 9 79.74 3.09 440.97 278.66 0.287  9b 8 33.62 1.62 202.13 174.98 0.025 12 12 51.89 4.79 435.12 232.48 0.115 18 10 6.26 4.48 498.31 658.74 0.053 20 10 21.82 2.06 228.39 180.24 0.396 22 11 40.13 9.95 995.26 756.98 0.136 23b 9 17.98 7.36 818.24 514.38 0.341 23a 10 39.29 7.06 882.82 883.16 0.075 26 12 4.75 5.34 485.51 168.09 0.179 ⁽*⁾Average correlation coefficient between adjacent marker pairs. ⁺Megabase

Linkage-linkage disequilibrium testing: Thirteen of the most significant LLD regions identified in Example 1 were selected for this phase of validation. Eight to twelve markers were genotyped in each region of interest. Marker haplotypes spanned physical distances of between 0.40-9.3 megabases (Mb) as they walked across genomic regions of interest. Chromosomal regions were analyzed in each of Data I and Data II. The criterion for validation of a LLD region to be considered validated was that the LRT was statistically significant at P<0.001 in both datasets.

Ten regions in both Data I and Data II showed positive results for the presence of QTL (FIG. 5). Regions on BTA12 and BTA18 showed significance in Data II, but not Data I. Regions on BTA 2 and BTA 9b showed significance in Data I, but not Data II. The eight remaining regions, corresponding to chromosomes 1, 3, 6, 9a, 22, 23(2), and 26, showed significant results at analogous locations within each region in both Data I and Data II.

Single-marker association testing: Three-hundred six markers representing all bovine chromosomes but BTA 17 were analyzed in the single marker association tests. The amount of marker representation on each chromosome was variable, and as expected, many marker associations surpassed the significance threshold (FIG. 5, FIG. 6). Of the previously genotyped markers analyzed, 65 and 94 were significant (P≦0.01) in Data II and Data I, respectively. For the previously ungenotyped markers tested for single-marker association, 37 and 10 markers were significant (P≦0.01) for Data II and Data I, respectively.

Agreement between single-marker association tests and LLD tests for the locations of QTL was not always consistent. Two and 18 significant single-marker associations (P<0.001) concur with the general vicinity of QTL peaks from the LLD results in each analysis of Data I and Data II, respectively. For Data I, the two significant single-marker associations were located on BTA22 and BTA26. For Data II, the 18 single-marker associations were located on BTA3, 9a, 12, 18, 22, 23b and 26. One single-marker association test was significant in both Data I and Data II, but the LLD analysis of Data I fell short of significance in that region.

Criteria necessary to be considered a validated single marker test included a significance level of P≦0.01 in Data II. In addition, phenotypic effects must have been of the same sign as had been previously discovered in the original analysis of the reduced Data I. Fifty-five markers in the previously genotyped group were validated, resulting in a 31% validation rate. A more stringent validation criterion was necessary for the marker associations in the previously ungenotyped group of markers. Twelve markers having effects of the same sign and a significance threshold surpassing P≦0.01 in both Data I and Data II were considered validated for association with twinning rate PTAs, and resulted in a 4.7% previously ungenotyped marker validation rate. Results of the marker validations are summarized in Table 2-2, where “effect” as used in the table is a regression coefficient that indicates the average change in twinning rate phenotype as one substitutes one allele for another. The phenotype in this case is twinning rate predicted transmitting ability (PTA).

TABLE 2.2 Single-marker association results for validated markers¹. Data II Data I SNP ID Mb* BTA Effect −log10(P) Effect −log10(P) Status⁺ rs29012841 3.70 BTA01 −0.024 3.1 −0.028 2.3 PG rs29013556 27.28 BTA01 0.023 2.7 0.029 2.2 PG rs29010972 28.34 BTA01 0.041 6.8 0.024 1.4 PG rs29009758 36.37 BTA01 −0.042 11.3 −0.022 1.7 PG rs29016850 115.88 BTA01 0.032 4.6 0.033 3.2 PG rs29012088 148.39 BTA01 0.022 2.4 0.051 5.6 PG rs29014793 27.76 BTA02 −0.024 2.8 −0.032 2.5 PG rs29010454 43.90 BTA04 −0.027 3.2 −0.034 2.4 PG rs29017408 46.41 BTA04 0.039 3.0 0.027 0.8 PG rs29010308 15.23 BTA05 0.021 2.1 0.028 1.9 PG rs29018610 55.07 BTA05 −0.023 3.0 −0.041 4.1 PG IGF1_SNP_3 66.79 BTA05 0.024 2.2 0.039 2.7 PNG IGF1_SNP_2 66.82 BTA05 −0.040 7.0 −0.045 5.3 PNG rs41257131 72.56 BTA05 0.042 7.4 0.029 1.6 PG rs29024681 8.48 BTA06 −0.055 9.8 −0.030 1.4 PG rs29024705 23.71 BTA06 −0.066 2.6 −0.076 4.2 PG rs29015348 44.44 BTA06 −0.033 4.8 −0.024 1.5 PG rs29018255 86.22 BTA06 0.065 5.5 0.059 3.1 PG rs29024685 86.64 BTA06 0.052 8.6 0.024 1.1 PG rs29024709 61.70 BTA07 −0.037 7.1 −0.001 0.0 PG rs29014932 62.91 BTA07 0.025 2.8 0.037 3.0 PG rs29027275 64.57 BTA07 −0.034 4.8 −0.006 0.2 PG rs29011411 68.12 BTA07 −0.063 18.1 −0.040 4.3 PG rs29012592 76.40 BTA07 −0.033 4.8 −0.023 1.0 PG rs29015209 55.72 BTA08 −0.034 3.5 −0.060 5.4 PG rs29020823 59.88 BTA08 −0.040 6.9 −0.036 3.6 PG rs29018125 62.79 BTA08 −0.045 8.8 −0.038 3.7 PG BTA_146334 33.62 BTA09 0.032 4.8 0.041 3.9 PNG rs29019730 89.50 BTA10 0.055 3.8 0.040 1.2 PG rs29025325 92.35 BTA10 −0.026 2.4 −0.026 2.0 PG rs29010503 46.70 BTA11 −0.022 2.4 −0.008 0.4 PG rs29010502 46.70 BTA11 0.022 2.4 0.008 0.4 PG rs29021155 66.09 BTA11 −0.047 9.0 −0.043 4.9 PG rs29026881 66.72 BTA11 −0.037 6.1 −0.032 2.8 PG rs41257406 100.98 BTA11 0.023 2.9 0.019 1.3 PG rs29025358 48.24 BTA12 0.029 3.3 0.048 3.8 PG rs29023435 9.62 BTA13 0.034 3.7 0.048 4.1 PG rs29023434 9.62 BTA13 −0.034 3.9 −0.056 5.5 PG rs29021895 13.78 BTA14 0.025 3.6 0.018 1.2 PG rs29025879 14.06 BTA14 −0.029 2.4 −0.036 2.2 PG NGS_74296 20.36 BTA14 0.040 6.6 0.032 2.4 PNG NGS_25065 20.73 BTA14 0.052 11.8 0.047 4.1 PNG rs29010471 20.97 BTA14 −0.047 8.5 −0.011 0.5 PG NGS_28234 21.24 BTA14 −0.051 11.1 −0.035 2.5 PNG rs29020900 21.76 BTA14 0.031 2.9 0.025 1.2 PG rs29010470 21.79 BTA14 0.048 8.8 0.030 2.2 PG rs29014046 27.73 BTA14 −0.031 4.4 −0.022 1.4 PG NGS_70865 31.98 BTA14 −0.027 2.8 −0.046 4.3 PNG NGS_38815 34.34 BTA14 0.026 3.6 0.040 3.6 PNG BAC_17596 35.00 BTA14 −0.036 4.7 −0.041 3.6 PNG BTB_005645 35.61 BTA14 0.037 5.1 0.039 2.6 PNG IBISS4snp1087 38.56 BTA14 0.037 6.6 0.043 4.7 PG AffyID3519 39.77 BTA14 −0.030 4.7 −0.044 5.1 PG rs29012557 40.07 BTA14 0.032 5.2 0.035 3.3 PG BTB_013878 40.24 BTA14 −0.058 13.4 −0.037 2.6 PNG rs41622614 43.40 BTA14 0.026 3.0 0.038 3.8 PNG rs29015662 23.04 BTA15 −0.032 4.3 −0.011 0.5 PG rs29021176 13.07 BTA18 −0.024 2.5 −0.009 0.4 PG rs29022856 41.27 BTA21 0.031 2.3 0.022 0.7 PG rs29020409 42.01 BTA23 −0.046 7.7 −0.033 1.6 PG rs29022783 51.04 BTA23 −0.053 10.3 −0.035 1.9 PG rs29015105 56.62 BTA24 0.067 4.9 0.067 4.6 PG rs29010042 12.73 BTA25 −0.035 2.8 −0.074 3.9 PG rs29010040 12.81 BTA25 −0.032 2.5 −0.070 3.7 PG rs29015664 23.04 BTA25 −0.034 4.4 −0.017 0.9 PG rs29021023 37.63 BTA26 0.031 2.5 0.031 1.8 PG rs29020955 8.76 BTA28 0.023 2.7 0.005 0.2 PG ⁺PG = Previously genotyped (Example 1); PNG = Previously not genotyped *Megabase ¹All PNG markers were previously found significant in Example 1. Sires used in the analysis of Example 1 are a subset of Data I.

Cross-validation: Sixty-seven markers (Table 2-2) entered the stepwise backward elimination analysis. Markers were allowed to exit the model if their significance value failed to exceed P<0.001. As expected, the marker IDs, along with the frequency at which each marker remained in the model across the ten data subsets was variable (FIG. 7). Thirty one markers were represented at least once. Markers not remaining in the 10 reduced marker panels for the training subsets are not represented in FIG. 7. Of the markers represented at least once, frequency of representation ranged from 1 to 10, with a median representation of 5. Eighteen markers in NM attained the criteria necessary to be included in the final marker panel (Table 2-3).

TABLE 2-3 Reduced marker panel used for predicting genomic breeding values. Genotype S.E. of Genotypic Parameter BTA Mb Genotypes¹ Estimates² estimates² Frequency³ Intercept 1.046 0.008 rs29010454 4 43.90 AA/AG/GG −0.010 −0.001 0.002 0.001 0.10 0.54 0.35 IGF1_SNP_2 5 66.82 AA/AG/GG 0.008 0.004 0.002 0.001 0.26 0.55 0.19 IGF1_SNP_3 5 66.79 AA/AG/GG 0.009 0.007 0.003 0.001 0.04 0.29 0.67 rs29015348 6 44.44 CC/CG/GG 0.010 0.002 0.002 0.002 0.57 0.35 0.07 rs29024681 6 8.48 AA/AG/GG 0.016 0.016 0.003 0.003 0.76 0.22 0.02 rs29012592 7 76.40 AA/AG/GG −0.007 −0.006 0.002 0.002 0.64 0.29 0.07 rs29011411 7 68.12 CC/CG/GG 0.006 0.004 0.002 0.001 0.09 0.49 0.42 rs29020823 8 59.88 CC/CT/TT 0.008 0.001 0.002 0.002 0.40 0.50 0.10 rs29015209 8 55.72 GG/GT/TT 0.013 0.011 0.003 0.003 0.65 0.31 0.04 BTA_146334 9 33.62 CC/CT/TT −0.012 −0.006 0.001 0.001 0.22 0.56 0.23 rs29010503 11 46.70 AA/AG/GG 0.003 −0.002 0.002 0.001 0.27 0.56 0.16 AffyID3519 14 39.77 CC/CG/GG −0.014 −0.019 0.004 0.004 0.24 0.51 0.25 IBISS4snp1087 14 38.56 AA/AC/CC −0.023 −0.002 0.004 0.002 0.26 0.53 0.21 BTB_005645 14 35.61 AA/AG/GG −0.011 −0.012 0.003 0.003 0.65 0.31 0.04 NGS_28234 14 21.24 AA/AG/GG 0.005 0.005 0.002 0.001 0.15 0.56 0.29 rs29015662 15 23.04 CC/CT/TT 0.006 0.002 0.002 0.002 0.39 0.52 0.09 rs29022783 23 51.04 AA/AG/GG 0.007 0.003 0.002 0.002 0.65 0.33 0.03 rs29020955 28 8.76 CC/CT/TT −0.010 −0.001 0.002 0.002 0.33 0.55 0.12 ¹Genotype categories 1 through 3 represented between hashes. ²Estimates (of difference in actual twinning rate among genotypes) reflect differences from the third genotype category, which is set to zero. ³Genotypic frequencies reflected in order of ‘Genotype’ category.

Efficacy of the reduced marker panel on predicting adjusted daughter averages was compared within each of the testing subsets. As previously mentioned, the markers and subsequently the coefficients predicted for each training subset, were variable. Total number of markers remaining in the model averaged 16.4 across the ten subsets in NM. Pearson correlation coefficients between marker-predicted and adjusted daughter averages in the 10 subsets from NM were calculated. Individuals having missing genotypes assume they are genotype 3, which is set to a zero value, and were subsequently removed. The average, median, and variance for the 10 correlation values obtained in NM were 0.418, 0.458, and 0.082, respectively.

Prediction Equations: Eighteen markers remained in the reduced marker panel. Four markers coincide near previously detected QTL peaks on BTA14 (Cobanoglu et al. 2005; Gonda et al. 2004). Two markers lie directly under the most likely position estimates from previous QTL detection on BTA5 (Cruikshank et al. 2004; Lien et al. 2000; Miewessen et al. 2002). One additional marker lays near previously detected QTL on BTA23 (Cruikshank et al. 2004). The remaining markers lie in locations not previously implicated in QTL detection, and include regions on chromosomes 4, 6, 7, 8, 9, 11, 15, and 28.

Genomic predictions of daughter averages for the bulls in Data I were calculated. Correlation of marker-predicted daughter averages with adjusted daughter averages, unadjusted daughter averages, and PTAs were 0.59, 0.14, and 0.34, respectively.

The majority of markers chosen for the reduced marker panel showed little association with dairy production traits. Marker rs29024681 located on BTA6 was significantly associated with protein percent (P<0.0001). No other marker association surpassed a significant threshold (P<0.001).

Example 3

The objectives of the study reported in this example were 1) to refine the location of the twinning or ovulation rate QTL on Bos taurus chromosome 5 (BTA5) as previously reported, by using a denser set of markers and a larger population along with a combined linkage-linkage disequilibrium analysis, 2) to test for evidence in the Holstein population of the QTL previously reported, and 3) to examine positional candidate genes.

Materials and Methods:

Animal resources: All animals used in this study were registered Holstein bulls from the United States and Canada. Twenty-five half-sib families were selected for use. Sires of the half-sib families had above average twinning rate values, as well as a moderate to large number of sons for which twinning rate predicted transmitting ability (PTA) had been calculated. A total of 358 bulls, including sires, with an average of 1,021 daughters per bull were genotyped. Two separate estimates of twinning rate predicted transmitting ability were used in analyses. The first dataset (Johanson et al., 2001; Data I) was based on daughter records obtained between 1994 and 1998, while the second dataset (Data II) used records obtained between 1998 and 2006. Twinning rate PTAs derived from Data II have not been described previously and are based on threshold model analysis of 2,718,434 Holstein calving records obtained from the Dairy Herd Improvement Association. The model for analysis of Data II included herd-year-season (with seasons January-June and July-December within each herd-year), age-parity (with up to 7 lactations and 2-mo age groups within each lactation), and sire (with relationships). A minimum of 10 calvings per herd with twinning rate>0.00% and <50% were required (mainly to eliminate herds that didn't report twinning). Overall frequency of multiple births was 4.06%. Triplets were included (coded as twins), though there were very few of these. Data I and Data II twinning rate predicted transmitting abilities were available for 342 and 291 sons, respectively. A total of 289 sons had predicted transmitting ability estimates from both data sets. Data I and Data II PTAs represent two substantially independent estimates of twinning rate phenotype for the bulls given that they are based on separate daughter records. The correlation between number of daughter records for bulls represented in both data sets was low (r=0.14). Predicted transmitting abilities of milk production, yields of protein and fat, percentages of milk protein and fat, productive life span, somatic cell score, and daughter pregnancy rate were obtained from the Animal Improvement Program Laboratory (AIPL) at USDA for examination of pleiotropic or correlated associations of candidate gene polymorphisms.

SNP Discovery: Seventeen genes were initially targeted for SNP discovery to increase marker density in selected areas of BTA5. PCR primer pairs (Table 3-1) were designed using Primer3 software (primer3-web 0.3.0; http://primer3.sourceforge.net/) and genomic sequence information from Build 3.1 of the bovine genome sequence.

TABLE 3-1 SNP discovery: PCR primers, amplicons and polymorphisms (the number following each sequence is its SEQ ID NO) Primer SNP Gene pair Forward primer Reverse Primer SNP location¹ IFNG 1 TTCAACTACTCCGGCCTAACTC (1) AGCAAGTGACTGAGTCCAAGAG (2) A/G 45363726 IFNG 2 TCAGTCCTAGAGAAGTCCCTTA (3) GTAATGAAGAACTCTGACACCC (4) A/C 45366254 PPM1H 1 CCATTCCCATCTGATTTTGAGT (5) GGGATTTTTCAAAGGACACTACA (6) T/C 50646545 PPM1H 1 CCATTCCCATCTGATTTTGAGT (5) GGGATTTTTCAAAGGACACTACA (6) A/T 50646565 PPM1H 2 ACCTGTCTCTGCAATTCAAAGTC (7) GTATTCTCCCTACACCGAAATCA (8) none INHBE 1 AATGGAAACAAGATTGGGACAG (9) TCCTTATCTGTCCTCCCACAGT (10) none INHBE 2 AATGTTCCTTCCAGTGGAGTTC (11) AAACTGTCCAAGAAGATGGGAG (12) none INHBE 3 GTGATTTTTCTCTGCCCTCTCC (13) TCTTGAAACTCCGACTGGACTG (14) none INHBE 4 ATTTGGGCCGGGTCTTAAGTTC (15) AAGCAGCAAATCCTTGAGGGAC (16) none INHBE 5 CCACCTGTGAGGACAGCAAAG (17) TACCTCAAGCTGCGTGACCAG (18) none INHBE 6 GACACGTATGATGCAAGAAGGA (19) CCATTTAAAGGCAGTGAGGAGT (20) none INHBE 7 GACTCAGTACCACCAGTGTCCA (21) TGGCTGGGGTATTTATAACCTG (22) none INHBE 8 AAAAGCCTGAGGAGCCTAGTG (23) GAGGCATTTGCTCTGAGATTG (24) none INHBE 9 TCTTCTCTCCCCAGACTTTCC (25) TGGCCTAGCAAACTAGCAAGA (26) A/G INHBE 9 TCTTCTCTCCCCAGACTTTCC (25) TGGCCTAGCAAACTAGCAAGA (26) T/C INHBE 10 AGGGTACAGAGCCAGATGGATA (27) TGCTCAATCTTCTCAAGGTCAA (28) none INHBC 1 CCTTCAACTTTACCCTCATCTC (29) CGTAGGAAGACTGAGATGACTG (29) T/C 54971414 INHBC 2 TGTAAAGCCTGGAATATGGTTG (31) GATCTTACGAAGTCTCTCTCCAC (32) none INHBC 3 CTGCCCACTAAATTCTCTACCC (33) AGCATCTTGGACAAGCTGCAC (34) A/G 54977605 INHBC 4 GATGATCTCATACTCCTGTCCCTC (35) ACATGACAATGACCTACAGTGTCC (36) none SNRPF 1 GAGCATGGTCTGTTCAGGTGGTA (37) ACTTGTCCATGCAGTTCAAACCT (38) A/T 58655355 SNRPF 1 GAGCATGGTCTGTTCAGGTGGTA (37) ACTTGTCCATGCAGTTCAAACCT (38) A/G 58655185 SYCP3 1 TGAGCACTGTTGAGATTTCCTA (39) GGTGGGGGTAAATCTTGAATA (40) none SYCP3 2 GTGCATGAGAGGATTTTGAGGGT (41) GGAAATCTCAACAGTGCTCAAAAC (42) none SYCP3 3 AATTCTTGAACCAGCTTTATCC (43) AAGTCCATTTTATCTCCAAGCA (44) none SYCP3 4 GAAAATACCATGAGCAGAGGAG (45) GCGACTAACACTTTCACATTTTC (46) none SYCP3 5 ATTTCTGTTGCTGGCAGTTTGG (47) GAAAGTGAAGTCGCTCAGTCGT (48) T/C 63550351 SYCP3 6 AGTCCATGGGGTCGCTAAGAGTC (49) AAAACACCCCAATAGCCGAGGA (50) C/G 63551475 SYCP3 7 AGAAATGATTCCTAACTTGCGTG (51) TTTTTCACCATCTTCCAGAGCTT (52) T/C 63553463 SYCP3 8 CACAGATATGAGTCCCGACCCTA (53) TGGGTGAAGGTTTCAGAGTGGT (54) T/G 63554668 SYCP3 9 ACAGGGTTGAACGTGAAATGGAG (55) CCCATAACTCCACTCACCAAAACA (56) A/G 63563991 SYCP3 9 ACAGGGTTGAACGTGAAATGGAG (55) CCCATAACTCCACTCACCAAAACA (56) T/C 63563950 SYCP3 10 TGGTTTTGAACTGTCGTGCTGG (57) CAGACTGAAACCCCCAAAACTCC (58) None IGF1 1 GGGCATAGACAAGATCCTTGACTAC (59) TGGTGATTGGCAAAGCTAGGATGTC (60) None IGF1 2 GAAATTTGATTCATGGTGTTCCCTC (61) AATTGTCCTCAAATTCTTAGCCACC (62) None IGF1 3 GCTGCCCTAAGAATGACAATAAACC (63) CACCTTTCATTTGATTCTGATTGCC (64) None IGF1 4 CTGAAACCACTTCTGCCACTTTACC (65) TGATGCTTGGCACTGTAAAGTGAGG (66) G/A 64222602 IGF1 5 AAGATGCCCATCACATCCTCCT (67) TGTGCCTTTCAGCGACTTTCAC (68) C/T 64271931 IGF1 6 GGGAATGACGTTCTGGTT (69) AACTGGAGAGCATCCACCAA (70) None IGF1 7 GTGCCACAAGTAGCAATGTGGA (71) GGCATGTGCATCCATTACTGACC (72) None IGF1 8 TGGAGGGTTTCCATGAACAGGT (73) TGTGGCTCAAGTCTCCATCCTT (74) A/G 64273280 IGF1 9 ATCTGAGAACATAACTCAGCAAA (75) AGAGGAGGTGAAAGCTTGATGG (76) C/A 64273948 IGF1 10 ATAATACCCACCCTGACCTGCT (77) CTCAGCAATACCTATCAGTCTCTAGC (78) None RIC8B 1 CTAGTACTGGGAGGCAGCAAGA (79) GCACATGGAAAGAGAACCAACA (80) Indef 65538446 RIC8B 2 CTAGTGCCCAGCACAGAGAAA (81) TTTGAGCTGCCAAAAGAGATG (82) T/C 65538923 RIC8B 3 CAGATGGCTAAATTGAATCAAC (83) CTAGCCCCACTCTTCACTTC (84) none RIC8B 4 TGTAATCAGTTTGCAAGTCTGG (85) GAGTTGCTTAAACCAATGGGAC (86) none RIC8B 5 CAACTGCCTCTCCTCAGCTAAT (87) AGACGTGTGACCTTGGGTAAGT (88) none RIC8B 6 GTAACAGTGCGTGTGAGAGGAG (89) GCAACCTATGGACTTGATTTGA (90) none RIC8B 7 TGTGCTTATGGAAGGAAGGTTT (91) TTCTGTGCCTTTGCTTGACTAA (92) none RIC8B 8 TGAAATGTGCATCAGGGTTAAG (93) TGTGCTTTGGGCAAGAGTATTC (94) none RIC8B 9 ACACCTTACTTGACCTGGAAAC (95) CAAGCCAACCTTACATTAAGAAC (96) A/G 65548378 RIC8B 10 CCGAGAAGGAAGCTCTGTAATC (97) GCCATTCCTGCTTTTTCAATC (98) T/C 65549208 RIC8B 11 GCCTCCAAATCTTAATTTTCTTCT (99) TCCCTTAACTCTGTAGTAGGAATG (100) none RIC8B 12 TCAGCACTGATCTCTCCAGTGT (101) CCACACATTTCCAAGCTAATGA (102) none CKAP4 1 AATGGCTACAGAAAATGCACAC (103) CAAGGGTTGTGCAAAAGTCTTC (104) A/G 66075291 SYN3 1 1 GCAAATGCAGACTGAATTGAAC (105) TGTGTGTACATGTGGGCATAAG (106) A/C 69267572 SYN3 1 1 GCAAATGCAGACTGAATTGAAC (105) TGTGTGTACATGTGGGCATAAG (106) T/C 69267450 SYN3 1 2 TCAGGAAGATCCCTTGGAGAG (107) TGAGAGAGGAGTGGACACTGAG (108) T/A 69276158 SYN3 1 2 tCAGGAAGATCCCTTGGAGAG (107) TGAGAGAGGAGTGGACACTGAG (108) T/C 69276119 SYN3 1 2 TCAGGAAGATCCCTTGGAGAG (107) TGAGAGAGGAGTGGACACTGAG (108) T/C 69276059 SYN3 1 2 TCAGGAAGATCCCTTGGAGAG (107) TGAGAGAGGAGTGGACACTGAG (108) A/C 69276054 SYN3 1 2 TCAGGAAGATCCCTTGGAGAG (107) TGAGAGAGGAGTGGACACTGAG (108) A/G 69275918 SYN3 1 3 CTTGTCCATCCTCCATCCTC (109) CAAAATCCTCAGCAAGGCAC (110) A/G 69280735 SYN3 1 3 CTTGTCCATCCTCCATCCTC (109) CAAAATCCTCAGCAAGGCAC (110) A/G 69280651 SYN3 1 3 CTTGTCCATCCTCCATCCTC (109) CAAAATCCTCAGCAAGGCAC (110) A/G 69280619 SYN3 1 3 CTTGTCCATCCTCCATCCTC (109) CAAAATCCTCAGCAAGGCAC (110) G/C 69280597 SYN3 1 4 TTGGGAGTGAGTGGAAGATGAG (111) AACCATCCCAGGGTATTTATGAG (112) T/C 69282937 SYN3 1 4 TTGGGAGTGAGTGGAAGATGAG (111) AACCATCCCAGGGTATTTATGAG (112) G/C 69282931 SYN3 1 4 TTGGGAGTGAGTGGAAGATGAG (111) AACCATCCCAGGGTATTTATGAG (112) T/C 59282905 SYN3 1 4 TTGGGAGTGAGTGGAAGATGAG (111) AACCATCCCAGGGTATTTATGAG (112) G/A 69282758 SYN3 1 4 TTGGGAGTGAGTGGAAGATGAG (111) AACCATCCCAGGGTATTTATGAG (112) G/A 69282744 SYN3 1 4 TTGGGAGTGAGTGGAAGAYGAG (111) AACCATCCCAGGGTATTTATGAG (112) G/A 69282695 SYN3 1 4 TTGGGAGTGAGTGGAAGATGAG (111) AACCATCCCAGGGTATTTATGAG (112) G/A 69282646 SYN3 1 4 TTGGGAGTGAGTGGAAGATGAG (111) AACCATCCCAGGGTATTTATGAG (112) indel 69282533 SYN3 1 4 TTGGGAGTGAGTGGAAGATGAG (111) AACCATCCCAGGGTATTTATGAG (112) T/C 69282448 SYN3 1 4 TTGGGAGTGAGTGGAAGATGAG (111) AACCATCCCAGGGTATTTATGAG (112) G/A 69282419 SYN3a 1 TGGTTAACTCATGTTGCTGGTC (113) AACAAAACCCACATGCCACC (114) T/C 69690399 SYN3a 1 TGGTTAACTCATGTTGCTGGTC (113) AACAAAACCCACATGCCACC (114) A/G 69690037 SYN3a 2 CGTCAGCCCATCAAGTATGTAAC (115) TTCAATAAATCCTGAGTGCCAGT (116) none SYN3a 3 GGTGAATTTGTCCCCAGAGAAC (117) CATGACAAGACTGGAGGGAAGA (118) A/G 69703470 SYN3a 4 TCGAGTCATTGGCATCCCTAGTA (119) CTTTCTTGACATTCCTGGGCTT (120) none SYN3a 5 TATAGAGGATAAAGCGGAGACAC (121) AAAGACTCACTTGCCCAATACC (122) none SYN3a 6 TCTGTAACTCGGATTGCTTTCTTG (123) GATTGCTGCTTACCAGTCTGTGT (124) none SYN3a 7 GTCGAATTGCATGATTGAAACC (125) GCGGAACCTTTTGTGGCTATTA (126) none TOM1 1 TGGATCACTGGCTGGGACACT (127) GTTCAGTAGGCGTGTAGTCCCAG (128) none TOM1 2 GCTGGCTTTGTCCTGTGAAGATG (129) CGGGAAGGCAGGTAGATTGACTC (130) none MCM5 1 GCTCTCCCTTTGCGACTTTCTCT (131) TCCAGCCTTCACAACCTACCACT (132) none MCM5 2 TCCTGGAATCGTGTGTGTGTGTG (133) AGGGCTAGAACCAGTGGAGAGGA (134) none HMCX1 1 ATAGCTCATGCGTGCCAGGACT (135) TCTGGCTACATCTTCTGGGCTCT (136) none HMCX1 2 CTGTGGATGACCTGATGGGAGTG (137) ACACAGCCACTTTCCCAACTACCT (138) none TXN2 1 CCAAACCACTGGGACAGTGAGT (139) GGAGGTGGAAGGGATCACATAA (140) none TXN2 2 GCGTCCAGTTCCCAGTTCCTT (141) GGCTTGGCTGGGTGTTACTGTC (142) A/C 73912042 EIF3S7 1 TTTGAGGAAGAGATCGTGTGTG (143) AGAAGTGAATGTGGCAAAAGAGT (144) T/C 73948028 EIF3S7 1 TTTGAGGAAGAGATCGTGTGTG (143) AGAAGTGAATGTGGCAAAAGAGT (144) A/G 73948017 EIF3S7 1 TTTGAGGAAGAGATCGTGTGTG (143) AGAAGTGAATGTGGCAAAAGAGT (144) A/G 73947882 EIF3S7 1 TTTGAGGAAGAGATCGTGTGTG (143) AGAAGTGAATGTGGCAAAAGAGT (144) A/G 73947851 EIF3S7 1 TTTGAGGAAGAGATCGTGTGTG (143) AGAAGTGAATGTGGCAAAAGAGT (144) G/C 73947448 EIF3S7 2 CCTATGTCGTTTGAATCACCAC (145) CCCCCTCCATTATAAAGTTTTC (146) T/C 73949685 EIF3S7 2 CCTATGTCGTTTGAATCACCAC (145) CCCCCTCCATTATAAAGTTTTC (146) T/C 73949318 EIF3S7 2 CCTATGTCGTTTGAATCACCAC (145) CCCCCTCCATTATAAAGTTTTC (146) T/C 73949144 SSTR3 1 GAGAATGGATGGATGGATGAAT (147) GCATTGCAGACAGATGCTTTAC (148) none SSTR3 2 CAGGCTGGTTATCATTCTGCTGTC (149) ATCTGTACCCCACTTCCAGCCTC (150) none SSTR3 3 GTTATCATTCTGCTGTCCCCTCT (151) TAACAAGATCTGTACCCCACTTCC (152) none SSTR3 4 CCACCCTCTTCCCTTCTAGATT (153) TTAGCTTAAGTGCCAGCTTTCC (154) none SSTR3 5 AATGTTGGAATCAGCCCTTAGA (155) GGGAAGAGGGTGGGATATAGAC (156) none SSTR3 6 AAGGTAACAACGCCTCCTTGTA (157) CATCATTTGGAAAAGGGAGAAG (158) none SSTR3 7 CACGTAGATGACCAGGGAGTTG (159) GGGAAGGACACAGTCTATCTCAG (160) none SSTR3 8 CACCAGGAAATAGAGGCCGAAG (161) GCATTCTGATCCCACTGGTCTAC (162) none SSTR3 9 ATCGAACCCTCATCTCCTGTG (163) GTGCTCAACATCATCAATGTGG (164) none SSTR3 10 TTGTGGTTCCCACATTTTCTCT (165) CTTGCTTTGTGTCCTATGGTTG (166) none SSTR3 11 AGGACTCTGGAAGGCAATACAC (157) TCCTCAGAGAAAATGTGGGAAC (168) none SSTR3 12 CAACAACCACATTGTACCAACC (169) GAGAGCTTGATCCAAAAGCAGT (170) none ¹Genomic location based on Bovine Genome Assembly version 3.1

PCR amplicons of approximately 800 bp containing exonic and intronic regions of the targeted genes were sequenced to identify polymorphisms (Table 3-1). PCR amplification was performed in a volume of 50 μl containing 1×PCR reaction buffer, 0.2 mM of each dNTP, 0.3 μM of each forward and reverse primer, 3% DMSO, 50 ng genomic DNA, and 1 unit of GoTaq DNA polymerase (Promega, USA). The DNA template used for SNP discovery was from a sire for which there was strong evidence of heterozygosity for a BTA5 twinning rate QTL (Cruickshank et al., 2004). Touchdown PCR was used in all amplifications with an initial denaturation at 95° C. for 2 min, followed by 10 cycles with denaturation at 94° C. for 45 sec, annealing for 30 sec, and extension at 72° C. for 70 sec. Annealing was at 65° C. initially, with a 1° C. reduction per cycle, down to 56° C. Twenty-five additional cycles were performed at the same denaturation and extension temperatures and times with annealing in all cycles at 56° C. for 45 sec. The last cycle was followed by a final incubation at 72° C. for 8 minutes. PCR products were purified with the MinElute PCR purification kit (QIAGEN, La Jolla, Calif.). Sequencing was performed in both directions using forward and reverse primers with the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA). Sequencing reactions were analyzed on an ABI3700 at the University of Wisconsin-Madison Biotechnology Center.

Genotyping: A total of 106 SNPs (Table 3-2) were successfully genotyped between 5 Mb and 85 Mb on chromosome 5. Assays were developed for an additional 14 SNPs but these failed to produce scorable genotypes. Of the 106 successfully genotyped SNPs, 71 had been used in a previous study (Kim et al., 2008) and were chosen considering marker location and minor allele frequency (MAF) in Holsteins. Minor allele frequency of all chosen markers was higher than 5% in the previous study (Kim et al. 2008). Thirty-six markers were newly developed within thirteen genes located between 55 Mb and 75 Mb on BTA5. Sixty-four of the 120 SNPs were located between 55 and 75 Mb on BTA5, to aid fine-mapping in this region.

TABLE 3-2 Location and identification of SNPs successfully genotyped. Position SNP ID Gene (Mb)¹ SNP ID Gene Position (Mb)¹ Table 3-1 IFNG 45363554 ss38322851 — 25542789 Table 3-1 IFNG 45366118 ss38333998 — 25612221 Table 3-1 PPM1H 50646348 ss38329202 — 25805367 Table 3-1 PPM1H 50646401 ss46526967 — 26051044 Table 3-1 INHBE 54966387 ss38331375 — 26213185 Table 3-1 INHBE 54966493 ss46526664 — 27484121 Table 3-1 INHBC 54977448 ss38334615 — 30959868 Table 3-1 SNRPF 58655362 ss38325441 — 31234560 Table 3-1 SNRPF 58655519 ss38332189 — 31393992 Table 3-1 SYCP3 63551646 ss38334249 — 32887508 Table 3-1 SYCP3 63553642 ss38324817 — 36286204 Table 3-1 SYCP3 63554840 ss38330986 — 37017623 Table 3-1 SYCP3 63564144 ss38322423 — 37772751 Table 3-1 SYCP3 63564196 ss38333459 — 38215050 Table 3-1 IGF1 64222809 ss38333457 — 38217081 Table 3-1 IGF1 64272121 ss38328259 — 39432441 Table 3-1 IGF1 64273448 ss38332923 — 39860001 Table 3-1 IGF1 64274119 ss38326081 — 43015339 Table 3-1 RIC8B 65538715 ss38333096 — 46763982 Table 3-1 RIC8B 65548214 ss38330228 — 49060290 Table 3-1 RIC8B 65549039 ss38332913 — 50736312 Table 3-1 CKAP4 66075473 ss38330728 — 50756220 Table 3-1 SYN3 69267636 ss38335703 — 53605874 Table 3-1 SYN3 69280766 ss38335704 — 53606067 Table 3-1 SYN3 69690202 ss38322168 — 58377370 Table 3-1 SYN3 69690568 ss38334842 — 58469225 Table 3-1 SYN3 69690601 ss38336375 — 58760185 Table 3-1 SYN3 69690919 ss38323314 — 59750361 Table 3-1 SYN3 69691212 ss38324210 — 60571988 Table 3-1 SYN3 69703674 ss38326045 — 62924050 Table 3-1 EIF3S7 73947622 ss38324469 — 63440624 Table 3-1 EIF3S7 73948047 ss38323954 — 64228962 Table 3-1 EIF3S7 73949317 ss38329400 — 64506317 Table 3-1 EIF3S7 73949530 ss38335844 — 65786585 Table 3-1 EIF3S7 73949897 ss38329473 — 66527964 ss38332405 — 5918850 ss38322176 — 66797547 ss38323163 — 6673215 ss38330735 — 67281572 ss38333589 — 8441967 ss38336504 — 67908996 ss38322192 — 8507929 ss38333147 — 69192069 ss38334594 — 8926891 ss46527005 — 69688243 ss38331238 — 10929300 ss46526764 — 69690399 ss38334462 — 11433635 ss46526677 — 69956055 ss38324419 — 12268428 ss38328460 — 74012901 ss38324418 — 12268601 ss38326324 — 74016524 ss38335775 — 12514696 ss38326323 — 74016632 ss38333537 — 12826659 ss38336298 — 74152696 ss38324813 — 16835901 ss38322350 — 76637809 ss38328726 — 17309176 ss38335160 — 78004851 ss38328727 — 17309229 ss38324385 — 78821368 ss38334640 — 18016855 ss38324387 — 78821694 ss38323327 — 19161398 ss38322778 — 84526473 ss38326471 — 22351578 ss38326000 — 87242524 ss38329587 — 25226289 ss38324928 — 87508710 ¹Based on Bovine Genome Assembly version 3.1.

Genotyping was performed by mass spectrometry of allele-specific primer extension reactions (Sequenom, San Diego, Calif.). Three microsatellite markers, CSSM022, ILSTS066, and BMS1216, located at 60.26, 60.27, and 69.3 Mb respectively, were also genotyped. DNA extraction and microsatellite genotyping were performed as described by Cruickshank et al. (2004).

Linkage, linkage disequilibrium, and haplotypes: Linkage map distances were estimated using CRIMAP 2.4 (Green et al. 1990). Linkage mapping was also used to examine the marker order predicted by the SNP genomic location obtained from bovine genome assembly 3.1. If the linkage map location was different (>30 cM) from its expected genomic position in genome assembly 3.1, it was considered as a probable genome assembly error.

Haplotype construction was performed using information on partial haplotypes obtained by linkage mapping. The most probable paternally inherited haplotype of offspring in each half-sib family was constructed using the chrompic option of CRIMAP. Sire haplotypes were inferred using reconstruction of paternally-inherited alleles and linkage phase in offspring obtained from CRIMAP.

LD was calculated as D′ and r² between all pairs of SNPs on chromosome 5 (BTA5). Hardy-Weinberg equilibrium (HWE) was tested for each SNP to find reduction in marker heterozygosity versus that predicted under HWE. To remove linkage effects within half-sib family from LD estimation, maternally-inherited haplotypes were analyzed in Haploview for construction of LD analysis (Barrett et al. 2005).

Linkage-linkage disequilibrium QTL mapping: Combined linkage-linkage disequilibrium analysis (LLD) was conducted to fine-map QTL. Linkage effects are based on transmission of alleles from sires to sons, while LD effects are estimated based on maternally-inherited haplotypes. There is increased probability that the QTL alleles carried on marker haplotypes with greater similarity are identical by descent (IBD). IBD probability between two haplotypes was estimated using the procedure proposed by Meuwissen and Goddard (2001). To predict IBD of a genomic position, eight-marker windows were used. Calculation of the likelihood at putative QTL positions was performed using restricted maximum likelihood (REML). The statistical model was

y=Zu+Wq+e  (1)

where: y is twinning rate PTA of the individual; u is vector of random polygenic effects; q is vector of additive QTL effects; e is vector of individual error terms; W is an incidence matrix of haplotypes relating phenotypic records to QTL alleles; and Z is a diagonal incidence matrix relating individual polygenic effects to individual sons.

The random effects u and q are assumed to be distributed u˜(0, Aσ_(u) ²) and q˜(0, Gσ_(q) ²), where: σ_(u) ² and σ_(q) ² are the polygenic variance and the additive QTL variance, respectively; A is the matrix explaining additive genetic relationship among animals; and G is the allele relationship (IBD) matrix between haplotypes.

To refine QTL location, Data I and II were analyzed both separately and together, in a multivariate analysis treating predicted transmitting abilities from the different data sets as separate traits. Individual random effects with corresponding variance and log likelihood ratio were estimated using the mixed procedure (SAS, 1996).

The log likelihood of the data under linkage only and LLD hypotheses was compared with that under the null hypothesis of no QTL at a given map position. The G matrix was constructed with linkage or LLD to test QTL hypotheses. The no QTL model with the A matrix accounting for additive genetic relationship is the same as the animal model (Lynch and Walsh 1998). The log likelihood test (LRT) was calculated as −2[(log likelihood of polygenic model)−(log likelihood polygenic with QTL model)], an approximation of the chi-square distribution with one (1) degree of freedom.

To determine the threshold level for a genome-wide scan, an analytical method proposed by Lander and Kruglyak (1995) was used. Comparison-wise error rates (CWER) of 2.3×10⁻⁵ and 7.2×10⁻⁴ yielded experiment-wise error rates corresponding to the significant and suggestive levels. For linkage QTL threshold, significant (p=5.0×10⁻⁵) and suggestive level (p=0.002) were used.

Association testing: A model that included the effects of sire and SNP genotype was used to evaluate the association between each SNP marker locus and a twinning rate PTA. A SNP genotype (or haplotype) effect and family effect were included in the linear model:

y=Xb+s+e  (2)

where: y is twinning rate PTA of the individual; b is a vector of the intercept and the regression coefficient; X is an incidence matrix of genotypes for b; and s is family effect. The size of vector b was variable for single SNP marker genotypes (2×1). The inverse of the prediction error variance (PEV) of PTA was used as a weight.

Positional candidate gene analysis: Based on initial mapping results, IGF1 was considered a strong positional candidate gene. This gene was screened for polymorphism over its full 72 kb genomic region, including 2 kb of flanking regions (5′ and 3′), using the same approach and sire as described above (Table 3-3).

TABLE 3-3 IGF1 SNP discovery: PCR primers, amplicons and polymorphisms (Number in parentheses after each sequence is SEQ ID NO). Primer PCR Start End SNP SNP Pair Forward Primer Reverse Primer Prod Base Base¹ (name) Location 1 TATTCCCACCATTCCAGAAAAC (171) GGCGAGCAATTTTATATTCCAG (172) 817 64278103 64277286 2 GAGTAGGCTTGAGATGGTCTTTT (173) TCAGATCCCACAGAATTGCATA (174) 816 64277365 64276549 C/T 64277279 (SNP1) 3 TTCGGCACTGGGAGTTATTTAT (175) TTTGCTGAGAATGGAAACTTGA (176) 774 64276604 64275830 4 ACTGACTTCGCCTTTCCAATAA (177) CCAGATCTTTTACAGCAGGTCA (178) 975 64275665 64274910 5 AAAGACACAGGCTCTTGCTCAAT (179) TCTGCACAGAAACAGAGAATGG (180) 829 64271572 64270743 6 CTGGCAATAAAGCAATTCTATCC (181) ACATTGTCACAGAGTGCTATAAACC 750 64270787 64270037 G/A 64270644 (182) (SNP2) 7 AAGGTTCTACATGGCAACTCTTA (183) ATTGGCTCAGACATGAAACAGA (184) 844 64270163 64269319 8 GACGAGACATTTAGATCCTCCCTA CAAGACTGGTTGACATGCTCTC (186) 757 64269381 64268624 (185) 9 CACAGCTTCACCAGACATTACAT (187) GAAGTTTAAATGCCTGCCAAAG (188) 854 64268703 64267849 10 CTCAGAAAACTGCTCCCAGAC (189) CAGCTGTCACACAAGTGCATAG (190) 793 64267936 64267143 11 TGGATTTGAGCTACACCATTTG (191) GTAGTCCTTGTTTTGGAGATGAG (192) 797 64267220 64266423 12 ACTTACTTGGTGGGTGGCATT (193) TACACTGCAACACTTTGGACCT (194) 786 64266502 64265716 13 GATCCAGAACCAAAGCAAGGTC (195) GGATCCAAAATGGAGCTATGAG (195) 769 64265805 64265036 14 CAGTGCAGGTGCCTCTCAAC (197) TTACCTTTGGACAAATGCAGAG (198) 847 64265134 64264287 15 ATTACTTTATGCAATTGGCAAGAAC GACCTGAAGTGGATTTGGAATG (200) 789 64264374 64263585 (199) 16 ACCATACACTTGGCCTTTCAAT (201) GAAGTCAATCAAACATGGTGCT (202) 841 64263639 64262798 17 TCAGCCTCAGCACTATTGACAT (203) AGTGGCTTCTCCAAGAGCATAC (201) 829 64262902 64262073 18 CCACCAGAAAGCAGAAACAGAT (205) TGTGATAAGGAAGCAGTTGAGG (206) 769 64262148 64261379 19 GAGCATGGCTTTGGTGAAATA (207) AGTGAGAACACTGAACCAGATTG (208) 756 64261386 64260630 20 CACAAGTGCTITCTCCTTACCA (209) TCTTCATTCCATGAGTCACCAC (210) 804 64260720 64259916 21 AAAGTTTCCAGCCGTTCATAGA (211) TGGTGGTTGAGAGCAACATTAC (212) 820 64259983 64259163 22 AAATCTGGCTGGCATTTCTG (213) AGGAGTTCCAATTTCCAGTTCA (214) 846 64259272 64258426 23 ATAGAACTGCTCCACGCATAAC (215) CCAATAATCCCATTTCAGAAGC (216) 787 64258489 64257702 24 ACGAGTCTTCAAACCTGTCTCC (217) ATGCATAAAGGTCCAAGTCCAG (218) 789 64257783 64258994 25 ATCACATCCCTCAATGTGGTTC (219) TGGTTTCGAGATGATTCAAGTG (220) 831 64257113 64256282 26 AGGGTGGAGCCGATGTAATAA (221) CACTATTGGACACAAAGCCATC (222) 832 64256357 64255525 27 CCCTCATCACTCACCAAAACTA (223) AGTAGACGGCATAAAGCATGAA (224) 761 64255592 64254831 28 TCCTCTAACAACACATAATCTAGCC GGGTACACAGTTGTCCAGAAGAC (226) 836 64254907 64254071 (225) 29 ACTCAGTGACTTCAGCAATGGA (227) CTGGACTAGGGGATACAGAGGA (228) 784 64254206 64253422 30 TCCATTACAAGTTCCACGCAAA (229) AGAGGAAGCTCTCAAGGAGGAT (230) 756 64253502 64252746 31 TACTGAGGTTGCAAGTGGGTTA (231) GGTAATAGAGCGTGGTGACCTT (232) 762 64252815 64252053 32 CCTCCCTGTGGTATTTTCTGTG (233) CTGGATAGCTGGCATCAAGAC (234) 791 64252338 64251547 33 CTAGGGTGAGGCGATGGAAA (235) TTGAGTTGCAAGATCCAAATGT (236) 797 64251626 64250829 34 AGTGGGATCATTCCTGTGAAAC (237) GAGGTCTGGGAAATGTCAAGG (238) 802 64250905 64250103 35 CCCTTTCAGGGAAATCTAGGAC (239) TGGTAAGGGATTAGGATCTCACA (240) 766 64250213 64249447 36 TTGGCTGCAAGGTATGTGAG (241) TGAACCTGGAATTGTGTTCTTG (242) 756 64249484 64248728 37 AAAACATTTGGAAAAGCACGAT (243) GAACTATCTCTGCATTGAGCAC (244) 848 64248793 64247945 38 AAGGATGTCAAACAAACAAGCA (245) TCCTTAATACCTTCCCAGGACA (246) 659 64248079 64247420 39 TGATCATGTAGAAGGGAAAGCA (247) TTTCACCATAAGCGGAAACATA (248) 845 64247260 64246415 40 ATCAGAGCAAGAGGGACTTCAC (249) GTTGCAGAATAGAGGAGGCAGT (250) 786 64246402 64245616 41 GGAGATAGAAGAACCACGGAAT (251) CAGCAGCAGCAGACTGAAAC (252) 751 64245791 64245040 42 CCCTCTTATCCCTGTGAAACTG (253) TCCTTCCTTAAAAGACCACCAA (254) 772 64245128 64244358 43 AGCATTCCAGTCAGCAAGATTT (255) TGAAATTGCTCAAGAAACATGG (256) 821 64244422 64243601 44 CTGGCTTCGCTCCAGATTTTA (257) TTAGCAGCAGCAAGACCATTTA (258) 814 64243890 64243076 45 TTTAGTGCTTGGACAGACCTGA (259) AGCTGACAGATTTCCCAATTTC (260) 814 64243150 64242336 46 AAAGCTGGAGACAGAACAGTCA (261) AACTCCTCTGGCAGGTTATGAA (262) 767 64242419 64241652 47 CCTACTAGAGAGTAAGGGTGACTTG AATGTAGGATGACAACCATCTGC (264) 763 64241735 64240972 A/G 64241296 (263) (SNP3) 48 GAATCAAAGATGGAAGGAAAGC (265) GGGTATTGGGAGAAGTGAGTGA (266) 827 64241065 64240238 49 ATGCTTGAGAATCTTTCCCTG (267) AGACCCCTGCTAAATTGTAAC (268) 878 64240368 64239490 50 GGTAGATTCCCCAAGGAGAAAG (269) CCTTGGACTCCTATTTTCCAGA (270) 815 64239855 64239040 51 CAGGATACAGTTGCCTTAGTTCC (271) AAAACATCAGAGGGAAAATGAC (272) 795 64239105 64238310 52 GACCCCTCAACACAGAAGTACC (273) ATTGTCAGGTGGGTATTCAAGC (274) 804 64238400 64237596 C/T 64237899 (SNP4) 53 GATCCTTGGGAAGTCTGTTGTC (275) ACCAACTTCAGGTTCACATGC (276) 814 64237670 64236856 G/A 64236951 (SNP5) 54 CACAGCCTGAGAAGCACTGATA (277) CCCATAAGGAGAGGAGGAGTTT (278) 750 64236974 64236224 55 TCAGAAGGATCATGAGAGCAGA (279) ACCAGACTCTTGCTATCCCAAC (280) 811 64236288 64235477 56 CATGCATCTGGGTGAGAACTAA (281) TGCAAGTTTCTCTGGAGATTGA 282) 810 64235551 64234741 G/A 64235335 (SNP6) 57 CAAAGGCATCCATCTGTTCTAA (283) GTCTTCCTCTICCTCCTCCTTT (284) 756 64234839 64234083 58 TGGTTCATTGAGAAGATACAGGA (285) GGGACCTCCCAAGAAAGAAT (286) 788 64234180 64233392 59 GAGCTAATCATGGTGGCTCAGT (287) CGGCTCATCTCCAAGCTAAAAG (288) 814 64233527 64232713 A/G 64232988 (SNP7) 60 GAGGTTTGGAGATAACAGACATGA GCTGAATGTTTCCTTTGGAGAG (290) 810 64232789 64231979 (289) 61 CATCAAGGAAGCCCAAGAATAC (291) AATTAGGAGGCTGAGAAAGGTC (292) 835 64232149 64231314 62 TCCTCATGCCTCAAAACCTTTA (293) TGCCATATGCAGAGAGGAAATA (294) 799 64231419 64230620 63 GGAAGCCAATGTTGTTTTCAG (295) ACCACCACGTTATCTCCCATAC (296) 793 64230685 64229892 64 CTCTCTTCCCACATCTGAAAGG (297) TTTCTCCCAAATAATCCACTCAC (298) 811 64229958 64229147 G/T 64229843 (SNP8) 65 CTACACCTATCCCATGCCTTGC (299) ATACCAGTGAAGGAGGCAACAT (300) 779 64229247 64228468 66 AAGTGGCAGATTCCAGATTTGT (301) GTTTGCTGGTCCCACTACTTCT (302) 838 64228540 64227702 67 AACAGGGCCACAATTCCTAGA (303) GAAAGATGCCTTCAGTGCTGTT (304) 753 64227766 64227013 68 CAATGCATGAAAGTTGTGTGTG (305) GGGCTATTCCTAGAAAATTCACCT 761 64227102 64226341 C/T 64226917 (306) (SNP9) 69 GTTGGTGTAGGAAACCTGTCTG (307) AAGAGACAGACATAGAGGACAAAC 762 64226493 64225731 C/T 64226066 (306) (SNP10) 70 CGACTTTCTCCCTTGGTATACAT (309) CTCTGGTCATTCATCATTGGTT (310) 836 64226067 64225231 71 ATGTAGCACCAATAAAGGCAGT (311) AAACTCATCGAGGCCAACTAAA (312) 830 64225294 64224464 72 GGTTTGAGCATCTTCAGGAACT (313) AGGCTTGATTTCTGGTTCTACA (314) 790 64224607 64223817 73 GGCCTCTCAATTGGTGAGAAA (315) CATCATCCACCACAGGTAACAA (316) 898 64223890 64222992 C/T 64223363 (SNP11) 74 CCTGAGAGGTCCATCTTTTCTG (317) GCAAAGGTGTCATTAGGAGCTT (318) 860 64220462 64219602 C/T 64220270 (SNP12) 75 CAAAACTAGCATTGTGCAGAGG (319) AGCTGGCCTACTTTTCTTCCTT (320) 808 64219666 64218858 76 CGAGAGAGAAGCAGACAGAAAG (321) CAATTCCAAGTGGAGGAAAGAG (322) 760 64218929 64218169 77 AAGTCGGAGTATTGGCAGGAT (323) AGTCCATGTTGATTTTGTGCTG (324) 802 64218254 64217452 78 AAGTCGGAGTATTGGCAGGAT (325) TGCAGGTTACTCTCAGGTGGTA (326) 768 64217526 64216758 G/C 64216781 (SNP13) 79 TGGAACCAGCCTCAGGTATAAG (327) CGGAGATTCCATGTAGCCTAGT (328) 849 64216832 64215983 80 AGTAGCACTGTGTGTGTGTTAGAT ATTGCATTATTTTCCCAAGGAC (330) 770 64216069 64215299 A/G 64215386 (329) (SNP14) 81 CTGGTTTCAGACCCTGGCTA (331) TCCACCCTTACATCCTAACAGC (332) 821 64215364 64214543 82 GCCTCTCACAAGCATTTGATCT (333) ATGAGTATCAGGGAATGGTGCT (334) 840 64214642 64213802 83 AGATCTGCAAGGTCAAAATCA (335) TTTCTCCCAAAACTATTTGCTG (336) 786 64213879 64213093 T/C 64213395 (SNP16) 84 AATTCCAAATATCCTTGGTGGT (337) TGGAAGGCTTTTCTGAACTTTT (338) 809 64213160 64212351 85 CACGTCAATCTCAAACAGATGC (339) TATCCTGTCTTGGCAATTCCTT (340) 850 64212435 64211585 86 TTCAAACAGAGTCAGGTGGTATC (341) GCAGGTAGCATAGTCCCTTCA (342) 755 64211725 64210970 A/T 64211268 (SNP17) 87 TTTAACAAGCTTTTCCTGGTTC (343) ATGTGGTTAGAGTGGGAAAGGA (344) 836 64210964 64210128 88 CAAAGATGGCCAATAAAGAAGTC (345) TTTAGGGAGGAAGCATTGATGT (346) 812 64210290 64209478 89 GTTTGTAGTGGGTGGAGATGGA (347) AACCTCAGGAGATGACGTTGTT (348) 796 64209544 64208748 90 TTGTTCCTATGTGACCTCAAGC (349) CATCTGTTGCTTTGCTCATTGT (350) 751 64208840 64208089 C/A 64208267 (SNP18) 91 CTTCTAGCAGGCCTCCACTTG (351) TAGACCCTGCTGATGGTATATG (352) 802 64208171 64207369 92 CATGTGCTTTCTTGTTGCTTGT (353) TTGACTCAGACCCATAGGGATT (354) 750 64207611 64206861 93 TTGATCACTCTAATGTTCCAGGT (355) TTGGATTAAACATTCTGGGTTC (356) 755 64206938 64206183 94 CCTTGGATAACAGAACTGGTTG (357) CCCATCTCTTTCTAACATTGGAT (358) 814 64206256 64205442 95 ACCCTGGCCCAAGTTGTCTAT (359) CTGGTGTGCTACTTAACTGCTTG (360) 750 64205523 64204773 96 GTTCCTCTATCCCACTCCCTTC (361) TTAATCCTCATACCACCCCTGT (362) 835 64204863 64204028 97 GAAACAAACATCTGACGACTCTG (363) TCTTGGGCTTAAAGAGAGGAAA (364) 851 64204173 64203322 98 CACCTGGGAAACCAAATCTAAT (365) TTATCTCAACCCACAGAACCCAT (366) 849 64203421 64202572 99 GAATTCAGCACCTCCAGCAT (367) TGAGTGGATTCTGATGGAAAGA (368) 826 64202694 64201868 G/A 64202520 (SNP19) 100 CCACTCCCAATTCCTCAAATAG (369) TTCTGCCATTTGCCTATAAGGT (370) 809 64201971 64201162 ¹Genomic location based on Bovine Genome Assembly version 3.1

A total of 848 Holstein bulls, which included 293 of those described above, were genotyped for IGF1 SNP association testing. SNPs were genotyped using a competitive allele-specific PCR system (KBiosciences, Hoddesdon, UK). PTA estimates were determined in separate analyses based on Data I and Data II, respectively. Twinning rate PTAs were available for 309 bulls from Data I (IGF1-Data I) and all 848 bulls from Data H (IGF1-Data II). Association tests were performed as described above. Single marker associations on BTA5 are shown in Table 3-4.

TABLE 3-4 Single marker associations on BTA5. Position Data I Data II SNP ID (Mb)¹ −log₁₀ (p) Effect −log₁₀ (p) Effect ss38332405 5.92 2.24 −0.03 2.94 0.02 ss38334594 8.93 0.76 0.01 8.24 −0.06 ss38324419 12.27 0.15 0.00 4.04 −0.04 ss38324418 12.27 0.94 −0.01 4.86 −0.04 ss38333537 12.83 0.18 0.00 2.78 −0.03 ss38324813 16.84 2.51 0.02 3.36 −0.03 ss46526664 27.48 0.02 0.00 3.64 −0.02 54966387² 54.97 1.06 −0.02 3.83 −0.06 54966493² 54.97 0.60 −0.01 5.30 −0.05 54977448² 54.98 0.47 −0.01 3.49 −0.06 ss38322168 58.38 3.76 −0.03 0.19 −0.01 58655362² 58.66 0.11 0.00 4.68 −0.04 63551646² 63.55 1.28 −0.02 3.02 −0.03 63554840² 63.55 0.39 −0.01 4.65 −0.05 64273448^(2,3) 64.27 4.89 −0.02 0.46 −0.01 65538715² 65.54 0.42 −0.01 4.05 −0.07 ss46527005 69.69 0.05 0.00 3.30 −0.02 ss46526677 69.96 4.38 0.03 0.77 0.01 ss38322778 84.53 0.02 0.00 3.48 0.01 ¹Physical position on the bovine genome assembly 3.1 ²Base pair locations of SNP corresponding to Table 3-1.

Results:

A total of 58 polymorphisms were discovered in 65,429 by sequenced across 17 genes in the initial SNP discovery effort. Four of the seventeen genes screened yielded no polymorphisms. The analysis revealed varying levels of polymorphism in the remaining 13 genes. The results for SNP discovery ranged from 21 SNPs and an indel found in 2934 by of the SYN3 genomic region, to zero SNPs found in 9100 by of the SSTR3 genomic region. One to eight (1-8) SNPs per targeted gene were arbitrarily chosen for subsequent genotyping.

Of 120 SNPs chosen for genotyping, 106 were successfully genotyped. Linkage map position of SNPs was compared with physical genomic position of SNPs on BTA5, and there was no disagreement in SNP order. SNPs used in this study spanned 81.6 Mb from 5 Mb to 86.6 Mb on BTA5. The average linkage map distance between markers was less than 1 cM for the 106 SNPs. No recombination was detected in 66 adjacent SNP marker pairs, primarily SNP pairs within genes. In these cases SNP order was inferred from bovine genome assembly 3.1. Total linkage map distance covered was 92 cM using the current data, or 78 cM based on the USDA third generation bovine linkage map (NCBI Map Viewer).

Linkage map distance between adjacent SNP markers was also compared with LD (r², D′) between adjacent markers. Although LD extended in some cases up to 8 cM, LD was typically decayed by 2 cM, based on r²≦0.2. At least one historical recombination or mutation was discovered in 94% of adjacent marker pairs that did not have any recombinants within family. Long-range LD (r²>0.5) was discovered between genomic regions including SYN3, at 69 Mb, and EIF3S7, at 74 Mb (FIG. 8). Between the genomic regions corresponding to SYN3 and EIF3S7, mean r² among all possible SNP pairs was 0.45, and mean D′ was 0.61. As only one intervening SNP was genotyped, the LD pattern between 69 and 74 Mb could not be adequately described.

The MAF range of SNPs was between 0.03 and 0.49. HWE tests detected eight SNPs in violation (p<0.05) of HWE, of which five deviated highly significantly from HWE (p<0.01) because the heterozygosity observed was lower than that predicted from allele frequencies. Two SNPs at 64 Mb, located within the IGF1 coding region, and another two SNPs, at 66 Mb and 67 Mb, respectively, also showed significant deviation.

The pattern of LD on BTA5 was surveyed prior to association testing and LLD analysis. Mean r² and W between adjacent marker pairs were 0.16 (r²) and 0.41 (D′), respectively. LD (r²) within eight-marker windows varied from 0.03 to 0.71. Mean LD of each haplotype window was calculated as r² or D′ between adjacent loci within eight-marker windows of the maternally-inherited haplotype. Less than fifty markers were genotyped between 5 and 55 Mb, a lower density than used for the genomic region between 55 and 75 Mb. Mean strength of LD (r²) within eight-marker windows in the genomic region on BTA5 between 0 and 55 Mb was less than 0.2, while in the region between 55 Mb and 75 Mb, more than 50% of eight-marker windows were in moderate LD (r²>0.4).

Evaluation for QTL affecting twinning rate resulted in multiple locations of interest on BTA5. The log-likelihood ratio (LRT) was plotted for a QTL located at the midpoint of each eight-marker window. Using Data I, the most significant LLD peak was at the midpoint of the SNP markers between 69.92 Mb and 69.99 Mb A 2 LOD-dropoff support interval for QTL location extended from 63 Mb to 70 Mb. LLD also detected a potential QTL at 25 Mb and at 50 Mb in the 5 to 60 Mb region on BTA5 using Data I (FIG. 9A). Using Data II, LLD detected a potential QTL at 30 Mb on BTA5 (FIG. 9B). A peak at the 69 Mb regions detected using Data I was also observed using Data II, though a higher peak was observed at approximately 64 Mb, corresponding well to the location of IGF1. Multivariate analysis using Data I combined with Data II (FIG. 9C) showed the strongest evidence of QTL in the 25-35 Mb and 64-75 Mb regions, with the highest significance at 64 Mb.

Using linkage mapping alone, weak evidence of QTL (p=0.02) was detected at the 64 Mb region using Data I (FIG. 9A). This region was spanned by microsatellite markers ILSTS066 (74 cM) and BMS1216 (78 cM). The QTL was also detected by linkage mapping in the 60-70 Mb region using Data II (FIG. 9B). Segregation of a twinning rate QTL was previously identified within two North American Holstein families (Cruickshank et al. 2004) between 74 and 78 cM, corresponding to 64 to 70 Mb.

Regarding individual markers, one significant association (p<2.3×10⁻⁵) and two suggestive associations (p<7.2×10⁻⁴) with twinning rate were observed between 5 Mb and 85 Mb on BTA5 using Data I. LD among these three SNPs was very weak (r²˜0.002) or slight (r²<0.2). Using Data I, two significant SNP associations were located, one each within the IGF1 (64.2 Mb) and the COL10A1 (69.9 Mb) gene (FIG. 10A). The SNP in COL10A1 was located within 1.5 Mb from the maximum QTL peak detected by LLD for Data I (FIG. 10A). None of the SNPs between 5 Mb and 55 Mb on BTA5 were significantly associated with twinning rate using Data I (FIG. 10A). In contrast, significant (p<2.3×10⁻⁵) single marker associations were observed in this region using Data II (FIG. 10B). This analysis produced five significant single marker associations with twinning rate (p<2.3×10⁻⁵). Two such SNPs were located around the 5 Mb region. Three other SNPs were detected near the 60 Mb region on BTA5, within the genes INHBC, INHBE and SNRPF, respectively (FIG. 10B). None of these SNPs were significantly associated with twinning rate PTA in Data I (p<0.01). A total of sixteen suggestive associations (p<7.2×10⁻⁴) were detected using either Data I or Data II; however, no suggestive associations in one data set were supported in the other data set at even a nominal p<0.05 level.

IGF1 was chosen for further examination as a candidate gene based on LLD results, single marker association results, and a known contribution to folliculogenesis (reviewed by Monget et al., 2002). Initial SNP discovery work focused on coding regions and revealed four SNPs. Additional SNP discovery efforts that completely covered the 72 kb region spanned by IGF1 revealed an additional eighteen SNPs (Table 3-3). Thirteen of these additional SNPs were successfully genotyped in the expanded set of bulls (Table 3-5), and none had genotype frequencies departing from expectation under Hardy-Weinberg equilibrium.

TABLE 3-5 Single-marker association tests of IGF1 SNP with twinning-rate predicted transmitting ability in the candidate gene analysis. SNP Minor allele IGF1-data I (n = 309) IGF1-data II (n = 848) identification Location, bp frequency Effect P-value Effect P-value SNP2 64270644 0.46 0.035 0.003 0.039 1.05 × 10⁻⁶ SNP3 64241296 0.21 −0.025 0.082 −0.026 0.005 SNP4 64237899 0.21 −0.026 0.075 −0.023 0.018 SNP5 64236951 0.39 0.052 2.66 × 10⁻⁶ 0.016 0.034 SNP9 64226917 0.22 −0.029 0.063 −0.003 0.727 SNP10 64226056 0.36 0.034 0.013 −0.021 0.014 SNP11 64223363 0.03 −0.057 0.24  −0.032 0.135 SNP12 64220270 0.36 0.061 2.75 × 10⁻⁷ 0.012 0.1 SNP14 64215386 0.36 −0.052 1.33 × 10⁻⁴ 0.019 0.029 SNP16 64213395 0.36 −0.056 2.95 × 10⁻⁵ 0.022 0.009 SNP17 64211268 0.15 0.019 0.282 −0.022 0.038 SNP18 64208267 0.36 0.057 3.17 × 10⁻⁵ −0.021 0.013 SNP19 64202520 0.44 0.059 9.14 × 10⁻⁷ 0.015 0.048 ¹Effects are expressed in the underlying threshhold model liability scale, which has no units. An effect of 0.03 would correspond to an increase in twinning rate of approximately 0.6%.

Bovine IGF1 consists of four exons of 60, 182, 157, and 63 bp. The size of introns 1, 2 and 3 are 4475, 51274 bp, and 15229 by respectively. Of the thirteen successfully genotyped SNPs, seven SNPs were located in intron 2, five were located in intron 3, and one was located very close to the 3′ UTR region. No SNPs were discovered in IGF1 exons.

A consistent, significant association with twinning rate PTA (p=0.003 and p=1.05×10⁻⁶, for IGF1-Data I and IGF1-Data II, respectively) was identified for IGF1 SNP2, located in intron 2. The allele substitution effect of G to A for IGF SNP2 was associated with a reduction of twinning rate and the direction of the effect was consistent between the two data sets (Table 3-5). Likewise, SNPs 5 and 19 were also associated with twinning rate in both sets of data with a consistent sign of the effect. However, in both cases the association in IGF1-Data II was only nominal (p<0.05), and other SNPs in high LD with these two (SNPs 12, 14, 16 and 18) exhibited lesser significance or inconsistent effects. The remaining SNPs were either not significantly associated or had allele substitution effect estimates of opposite sign from the two data sets (Table 3-5). Associations of IGF1 SNP 2 with lactation or production traits was not significant (p>0.05). SNP2 was not located in a known regulatory sequence or a region highly conserved across species, suggesting it is a marker in high LD with the underlying, functional polymorphism.

Two haplotype blocks (Wang et al. 2002) were detected in IGF1 using the D′ measure of linkage disequilibrium (FIG. 11). Multiple haplotypes were observed within each block. One haplotype block spanned SNP2, 3 and 4 in intron2 (Haploblock1) and another haplotype block extended from SNP5 to SNP19 in introns 2 and 3 and the 3′ downstream region (Haploblock 2) of IGF1. Haploblocks 1 and 2 are distinguished by historical recombination in the middle of intron 2.

REFERENCES

-   Abel K., Reneland R., Kammerer S., Mah S.,Hoyal C., Cantor C. R.,     Nelson M. R. & Braun A. (2006) Genome-wide SNP association:     Identification of susceptibility alleles for osteoarthritis.     Autoimmunity Reviews 5, 258-63. -   Allan, M. F., L. A. Kuehn, R. A. Cushamn, W. M. Snelling, S. E.     Echternkamp, and R. M. Thallman. 2008. Confirmation of QTL using a     low density SNP map for twinning and ovulation rate on bovine     chromosome 5. J. Animal Science (in press). -   Allan M. F., Thallman R. M., Cushman R. A., Echternkamp S. E.,     Kuehn L. A. & Snelling W. M. (2007) Fine mapping of QTL for twinning     and ovulation rate using low density SNP map in conjunction with     microsatellites marker information in the USMARC twinning     population. Plant and Animal Genome XV, San Diego, Calif. -   Andersson L. & Georges M. (2004) Domestic-Animal Genomics:     Deciphering the genetics of complex traits. Nature Review Genetics     5, 202-12. -   Arias J A. Kirkpatrick B W (2004) Mapping of bovine ovulation rate     QTL using three generation pedigrees. Animal Genetics 35, 7-13. -   Barrett J C, Fry B, Mailer J, Daly M J (2004) Haploview: analysis     and visualization of LD and haplotype maps. Bioinformatics 21,     263-265. -   Barrett J C, Cardon L R (2006) Evaluating coverage of genome-wide     association studies. Nature Genetics 38, 659-662. -   Beerepoot G. M., Dykhuizen A. A., Nielen M. & Schukken Y. H. (1992)     The economics of naturally occurring twinning in dairy cattle.     Journal of Dairy Science 75, 1044-51. -   Blattman A N, Kirkpatrick B W, Gregory K E (1996) A search for     quantitative trait loci for ovulation rate in cattle. Animal     Genetics 27, 157-162. -   Calus M. P. L., Meuwissen T. H. E., de Roos A. P. W. &     Veerkamp R. F. (2008) Accuracy of genomic selection using different     methods to define haplotypes. Genetics 178, 553-61. -   Chiu Y E, Liu S Y, Tsai Y Y (2005) A comparison in association and     linkage genome-wide scans for alcoholism susceptibility genes using     single-nucleotide polymorphisms. BMC Genetics 6, Supplement 1:589. -   Cobanoglu O, Berger P J, Kirkpatrick B W (2005) Genome screen for     twinning rate QTL in four North American Holstein families. Animal     Genetics 36, 303-308. -   Cruickshank J., Dentine M., Berger P. J. & Kirkpatrick B. W. (2004)     Evidence for quantitative trait loci affecting twinning rate in     North American Holstein cattle. Animal Genetics 35, 206-12. -   Draper N. R. & Smith H. (1981) Applied regression analysis. Second     edition. New York: John Wiley and Sons, Inc. -   Echternkamp S. E., Spicer L. J., Gregory K. E., Canning S. F. &     Hammond J. M. (1990) Concentrations of insulin-like growth factor-I     in blood and ovarian follicular fluid of cattle select for twins.     Biology of Reproduction 43, 8-14. -   Eddy R. G., Davies O. & David C. (1991) An economic assessment of     twin births in British dairy herds. Veterinary Record 129, 526-9. -   Ehrenreich I M, Stafford P A, Purugganna M D (2007) The genetic     architecture of shoot branching in Arabidopsis thaliana: a     comparative assessment of candidate gene associations vs.     quantitative trait locus mapping. Genetics 176, 1223-1236. -   Fricke P. M. & Wiltbank M. C. (1999) Effect of milk production on     the incidence of double ovulation in dairy cows. Theriogenology 52,     1133-43. -   Gilmour A R, Gogel B J, Cullis B R, Welham S J, Thompson R (2002)     ASReml User Guide Release 1.0. -   Goddard M. E., Hayes B. J., McPartlan A. & Chamberlain J. (2006) Can     the same genetic markers be used in multiple breeds? Proc. 8th     World. Congr. Genet. Appl. Livest. Prod. Belo Horizonte, Brazil. -   Gonda M G, Arias J A, Shook G W, Kirkpatrick B W (2004)     Identification of an ovulation rate QTL in cattle on BTA14 using     selective DNA pooling and interval mapping. Animal Genetics 35,     298-304. -   Grapes L, Dekkers C M, Rothschild M F, Fernando R L (2004) Comparing     linkage disequilibrium-based methods for fine mapping quantitative     trait loci. Genetics 166, 1561-1570. -   Green P, Falls K, Crook S (1990) Documentation for CRIMAP. version     2.4. -   Gregory K E, Bennett G L, Van Vleck L D, Echternkamp S E, Cundiff L     V (1997) Genetic and environmental parameters for ovulation rate,     twinning rate, and environmental parameters for ovulation rate,     twinning rate and weight traits in a cattle population selected for     twinning. Journal of Animal Science 75, 1213-1222. -   Grisart B, Coppieters W, Farnir F, Karim L, Ford C et al. (2002)     Positional candidate cloning of a QTL in dairy cattle:     Identification of a missense mutation in the bovine DGAT1 gene with     major effect on milk yield and composition. Genome Research 12.     222-231. -   Hampe J, Franke A, Rosentiel P, Till A, Teuber M et al. (2007) A     genome-wide association scan of nonsynonymous SNPs identifies a     susceptibility variant for Crohn's disease in ATG16L1. Nature     Genetics 39, 207-211. -   Hardenbol P., Yu F. L., Belmont J., MacKenzie J., Bruckner C.,     Brundage T., Boudreau A., Chow S., Eberle J. & Erbilgin A. (2005)     Highly multiplexed molecular inversion probe genotyping: over 10,000     targeted SNPs genotyped in a single tube assay. Genome Research 15,     269-75. -   Hayes B J, Chamberlain A, Goddard M E (2006) Use of linkage markers     in linkage disequilibrium with QTL in breeding programs. Proceedings     of the 8th World Congress of Genetics Applied to Livestock     Production. -   Hayes B J., Visscher P., McPartlan H. & Goddard M. E. (2003) Novel     Multilocus Measure of linkage disequilibrium to estimate past     effective population size. Genome Research 13, 635-43. -   Hayes B J, Goddard M E (2001) The distribution of the effects of     genes affecting quantitative traits in livestock. Genetics Selection     Evolution 33, 209-229. -   Hirschhorn J. N. & Daly M. J. (2005) Genome-wide association studies     for common diseases and complex traits. Nature Review Genetics 6,     95-108. -   Johanson J M, Berger P J, Kirkpatrick B W, Dentine M R (2001)     Twinning rates for North American Holstein. Journal Dairy Science     84, 2081-2088. -   Kappes S M, Benne G L, Keele J W, Echternkamp S E, Gregory K E,     Thallman R M (2000) Initial results of genomic scans for ovulation     rate in a cattle population selected for increased twinning rate.     Journal of Animal Science 78, 3053-3059. -   Karlsen A., Ruane J., Klemetsdal G. & Heringstad B. (2000) Twinning     rate in Norwegian cattle: frequency, (co)variance components, and     genetic trends. Journal of Animal Science 78, 15-20. -   Kirkpatrick B W, Byla B M, Gregory K E (2000) Mapping quantitative     trait loci for bovine ovulation rate. Mammalian Genome 11, 136-139. -   Lander E. S. & Kruglyak L. (1995) Genetic dissection of complex     traits: guidelines for interpreting and reporting linkage results.     Nature Genetics 11, 241-7. -   Lien S., Karlsen A., Klemetsdal G. et al. (2000) A primary screen of     the bovine genome for quantitative trait loci affecting twinning     rate. Mammalian Genome 11, 877-82. -   Lynch M. & Walsh B. (1998) Genetics and Analysis of Quantitative     Traits. Sinauer Associates, Sunderland, Mass. -   Markusfeld O. (1987) Periparturient traits in seven high dairy     herds. Incidence rates, association with parity, and     interrelationships among traits. Journal of Dairy Science 60,     158-66. -   Maestrini E., Pagnamenta A. T., Lamb J. A., Bacchelli E., Sykes N.     H., Sousa I., Toma C., Barnby G., Butler H., Winchester L.,     Scerri T. S., Minopoli F., Reichert J., Cai G., Buxbaum J. D.,     Korvatska O., Schellenberg G. D., Dawson G., de Bildt A.,     Minderaa R. B., Mulder E. J., Morris A. P., Bailey A. J. &     Monaco A. P. (2009) High-density SNP association study and copy     number variation analysis of the AUTS1 and AUTS5 loci implicate the     IMMP2L-DOCK4 gene region in autism susceptibility. Molecular     Psychiatry 1-15. -   Meuwissen T. H. E. & Goddard M. E. (2001) Prediction of identity by     descent probabilities from marker-haplotypes. Genetics Selection     Evolution 33, 605-34. -   Meuwissen T. H. E., Karlsen A., Lien S., Olsaker I. &     Goddard M. E. (2002) Fine mapping of a quantitative trait locus     twinning rate using combined linkage and linkage disequilibrium     mapping. Genetics 161, 373-9. -   Meuwissen T H E, Goddard M E (2001) Prediction of identity by     descent probabilities from marker-haplotypes. Genetics Selection     Evolution 33, 605-634. -   Meuwissen T H E, Hayes B J, Goddard M E (2001) Prediction of total     genetic value using genome-wide dense marker maps. Genetics 157,     1819-1829. -   Monget, P., S. Fabre, P. Mulsant, F. Lecerf, J.-M. Elsen, S.     Mazerbourg, C. Pisselet and D Monniaux. (2002) Regulation of ovarian     folliculogenesis by IGF and BMP system in domestic animals. Domestic     Animal Endocrinology 23:139-54. -   Nezer C, Collette C, Moreau L, Brouwers B, Kim J J et al. (2003)     Haplotype sharing refines the location of an imprinted quantitative     trait locus with major effect on muscle mass to a 250-kb chromosome     segment containing the porcine IGF2 gene. Genetics 165, 277-285. -   Nielen M., Schukken Y. H., Scholl D. T., Wilbrink H. J. &     Brand A. (1989) Twinning in dairy cattle: A study of risk factors     and effects. Theriogenology 32, 845-62. -   Rioux J D, Xavier R J, Taylor K D, Silverberg M S, Goyette A et     al. (2007) Genome-wide association study identifies new     susceptibility loci for Crohn disease and implicates autophagy in     disease pathogenesis. Nature Genetics 39, 596-604. -   Risch N, Merikangas K (1996) The future of genetic studies of     complex human diseases. Science 273, 1516-1517. -   Ron, M., E. Ezra & Weller. J. I. (1990) Genetic analysis of twinning     rate in Israeli Holstein cattle. Genetics Selection Evolution 22,     349-59. -   Sobel E., Papp J.C. & Lange K. (2002) Detection and integration of     genotyping errors in statistical genetics. American Journal of Human     Genetics 70, 496-508. -   Sobel E. & Lange K. (1996) Descent graphs in pedigree analysis:     applications to haplotyping, location scores, and marker sharing     statistics. American Journal of Human Genetics 58, 1323-37. -   Sved JA (1971) Linkage disequilibrium and homozygosity of chromosome     segment in finite populations. Theoretical Population Biology 2,     125-141. -   Thomas D C (2006) Are we ready for genome-wide association studies?     Cancer Epidemiology Biomarkers & Prevention 15, 595-598. -   VanRaden P. M., Van Tassell C. P., Wiggans G. R., Sonstegard T. S.,     Schnabel R. D., Taylor J. F. & Schenkel F. S. (2009) Invited Review:     Reliability of genomic predictions for North American Holstein     bulls. Journal of Dairy Science 92, 16-24. -   Wang, N., J. M. Akey, K. Zhung, R. Chakraborty and L. Jin. 2002.     Distribution of recombination crossovers and the origin of haplotype     blocks: the interplay of population history, recombination, and     mutation.

Wang W Y, Barratt B J, Clayton D G, Todd J A (2005) Genome-wide association studies: Theoretical and practical concerns. Nature Review Genetics 6, 109-118.

-   Weller J I, Song J Z, Heyen D W, Lewin H A, Ron M (1998) A new     approach to the problem of multiple comparisons in the genetic     dissection of complex trait. Genetics 150, 1699-1706. -   Windig J J, Meuwissen T H E (2004) Rapid haplotype reconstruction in     pedigrees with dense marker maps. Journal of Animal Breeding and     Genetics 121, 26-39. -   Zimin A. V., Delcher A. L., Florea L., Kelley D. R., Schatz M. C.,     Puiu D., Hanrahan F., Pertea G., Van Tassel C. P., Sonstegard T. S.,     Marçais G., Roberts M., Subramanian P., Yorke J. A. &     Salzberg S. L. (2009) A whole-genome assembly of the domestic cow,     Bos taurus. Genome Biology 10, 42.1-10.

In the specification, there have been disclosed typical preferred embodiments of the invention. Although specific terms are employed, they are used in a generic and descriptive sense only and not for purposes of limitation. The scope of the invention is set forth in the appended claims. Modifications and variations of the invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims the invention may be practiced otherwise than as specifically described. 

1. A collection of one or more polynucleotides, each of which is at least partially complementary to a sequence in a bovine genome, the presence or absence of which such complementary sequences in the bovine genome is quantitatively associated with the trait of twinning in a cattle population, wherein the collection comprises at least one sequence that is quantitatively associated with twinning with statistical significance of at least p≦0.01.
 2. The collection of claim 1 wherein the collection comprises at least one sequence that is quantitatively associated with twinning with statistical significance of at least p≦0.001.
 3. The collection of claim 1 wherein each polynucleotide represents one allele of a SNP in the bovine genome.
 4. The collection of claim 1 wherein in at least one of the polynucleotides is complementary to a sequence located on bovine chromosome 5 (BTA5).
 5. The collection of claim 4 wherein at least one of the polynucleotides is complementary to a sequence located in a genomic sequence for Insulin-like Growth Factor 1 (IGF1).
 6. The collection of claim 1 wherein at least one of the polynucleotides is complementary to a sequence in a genomic sequence for synapsin III (SYN3), synapsin III isoform A (SYN3A), collagen type X alpha-1 (COL10A1) precursor, eukaryotic translation initiation factor 3, subunit 7 (EIF3S7), insulin-like growth factor 1 (IGF1), inhibin beta-C (INHBC), cytoskeleton-associated protein 4 (CKAP4), or Alpha2-HS glycoprotein (AHSG.)
 7. The collection of claim 1 comprising at least one polynucleotide useful for detecting one or more of the SNPs: NCBI ID Nos.: rs29012841, rs29013556, rs29010972, rs29009758, rs29016850, rs29012088, rs29014793, rs29010454, rs29017408, rs29010308, rs29018610, IGF1_SNP_(—)3, IGF1_SNP_(—)2, rs41257131, rs29024681, rs29024705, rs29015348, rs29018255, rs29024685, rs29024709, rs29014932, rs29027275, rs29011411, rs2901259, rs29015209, rs29020823, rs29018125, BTA_(—)146334, rs29019730, rs29025325, rs29010503, rs29010502, rs29021155, rs29026881, rs41257406, rs29025358, rs29023435, rs29023434, rs29021895, rs29025879, NGS_(—)74296, NGS_(—)25065, rs29010471, NGS_(—)28234, rs29020900, rs29010470, rs29014046, NGS_(—)70865, NGS_(—)38815, BAC_(—)17596, BTB_(—)005645, IBISS4snp1087, AffyID3519, rs29012557, BTB_(—)013878, rs41622614, rs29015662, rs290211.76, rs29022856, rs29020409, rs29022783, rs29015105, rs29010042, rs29010040, rs29015664, rs29021023, and rs29020955.
 8. The collection of claim 7, comprising one or more of SNPs: rs29010454, IGF1_SNP_(—)2, IGF1_SNP_(—)3, rs29015348, rs29024681, rs29012592, rs29011411, rs29020823, rs29015209, BTA_(—)146334, rs29010503, AffyID3519, IBISS4snp1087, BTB_(—)005645, NGS_(—)28234, rs29015662, rs29022783 and rs29020955.
 9. The collection of claim 4, comprising one or more of SNPs: ss38332405, ss38334594, ss38324419, ss38324418, ss38333537, ss38324813, ss46526664, 54966387, 54966493, 54977448, ss38322168, 58655362, 63551646, 63554840, 64273448, 65538715, ss46527005, ss46526677 and ss38322778.
 10. A method for estimating the likelihood of twinning in one or more members of a cattle population comprising the steps of a) providing a collection of one or more polynucleotides, each of which is at least partially complementary to a sequence in a bovine genome, comprising at least one sequence that is quantitatively associated with twinning with statistical significance of at least p≦0.01; b) using the collection to determine the presence or absence of sequences complementary to one or more polynucleotides from the collection in one or more members of the cattle population genome, wherein the presence or absence of the complementary sequences is quantitatively associated with the trait of twinning in a cattle population; c) estimating the likelihood of twinning based on the results of step b).
 11. The method of claim 10 wherein the estimating step b) comprises a laboratory analysis or a field test, and step c) comprises a statistical calculation.
 12. The method of claim 10 wherein the collection comprises at least one sequence that is quantitatively associated with twinning with statistical significance of at least p≦0.01.
 13. The method of claim 10 wherein at least each polynucleotide in the collection is useful for detecting the presence or absence of one allele of a SNP in the bovine genome.
 14. The method of claim 10 wherein in at least one of the polynucleotides in the collection is complementary to a sequence located on bovine chromosome 5 (BTA5).
 15. The method of claim 14 wherein at least one of the polynucleotides in the collection is complementary to a sequence located in a genomic sequence for Insulin-like Growth Factor 1 (IGF1).
 16. The method of claim 10 wherein at least one of the polynucleotides in the collection is complementary to a sequence in a genomic sequence for synapsin III (SYN3), synapsin III isoform A (SYN3A), collagen type X alpha-1 (COL10A1) precursor, eukaryotic translation initiation factor 3, subunit 0.7 (EIF3S7), insulin-like growth factor 1 (IGF1), inhibin beta-C (INHBC), cytoskeleton-associated protein 4 (CKAP4), or Alpha2-HS glycoprotein (AHSG.)
 17. The method of claim 10, wherein the collection comprises at least one polynucleotide useful for detecting one or more of the SNPs: NCBI ID Nos.: rs29012841, rs29013556, rs29010972, rs29009758, rs29016850, rs29012088, rs29014793, rs29010454, rs29017408, rs29010308, rs29018610, IGF1_SNP_(—)3, IGF1_SNP_(—)2, rs41257131, rs29024681, rs29024705, rs29015348, rs29018255, rs29024685, rs29024709, rs29014932, rs29027275, rs29011411, rs2901259, rs29015209, rs29020823, rs29018125, BTA_(—)146334, rs29019730, rs29025325, rs29010503, rs29010502, rs29021155, rs29026881, rs41257406, rs29025358, rs29023435, rs29023434, rs29021895, rs29025879, NGS_(—)74296, NGS_(—)25065, rs29010471, NGS_(—)28234, rs29020900, rs29010470, rs29014046, NGS_(—)70865, NGS_(—)38815, BAC_(—)17596, BTB_(—)005645, IBISS4snp1087, AffyID3519, rs29012557, BTB_(—)013878, rs41622614, rs29015662, rs29021176, rs29022856, rs29020409, rs29022783, rs29015105, rs29010042, rs29010040, rs29015664, rs29021023, and rs29020955.
 18. The method of claim 17, wherein the collection comprises comprising one or more of SNPs: rs29010454, IGF1_SNP_(—)2, IGF1_SNP_(—)3, rs29015348, rs29024681, rs29012592, rs29011411, rs29020823, rs29015209, BTA_(—)146334, rs29010503, AffyID3519, IBISS4snp1087, BTB_(—)005645, NGS_(—)28234, rs29015662, rs29022783 and rs29020955.
 19. The method of claim 14, wherein the collection comprises one or more of SNPs: ss38332405, ss38334594, ss38324419, ss38324418, ss38333537, ss38324813, ss46526664, 54966387, 54966493, 54977448, ss38322168, 58655362, 63551646, 63554840, 64273448, 65538715, ss46527005, ss46526677 and ss38322778.
 20. A kit for estimating breeding value or predicting the likelihood of twinning comprising: (1) a collection of polynucleotides according to claim 1; and (2) instructions for using the collections for estimating breeding value or predicting the likelihood or genetic potential for twinning. 